Compositions and treatments for inhibiting kinase and/or HMG-CoA reductase

ABSTRACT

The present invention provides compositions of matter, kits and methods for their use in the treatment of MAP kinase-related conditions and/or HMG-CoA reductase-related conditions. In particular, the invention provides compositions for treating inflammatory and/or cardiovascular conditions in an animal subject by inhibiting p38α MAP kinase and/or HMG-CoA reductase, as well as providing formulations and modes of administering such compositions. The invention further provides methods for the rational design of inhibitors of MAP kinase, HMG-CoA reductase, or both for use in the practice of the present invention.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationsSer. Nos. 60/567,118, filed Apr. 29, 2004 and 60/630,684, filed Nov. 23,2004. Such applications are incorporated by reference herein, for allpurposes. U.S. Provisional Patent Applications Ser. No. 60/630,683,filed Nov. 23, 2004, is also incorporated by reference herein, for allpurposes.

BACKGROUND

The pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α)and interleukin-1β (IL-1β), contribute to the pathogenesis of variousallergic, inflammatory and autoimmune diseases. Consequently, multipletherapeutic approaches have been aimed at reducing the expression and/oractivity of such pro-inflammatory cytokines. Examples of these includethe use of IL-1 receptor antagonists, TNF-α converting enzymeinhibitors, and inhibitors of certain enzymes that play a role in signaltransduction pathways associated with inflammation, including responsesto and expression of TNF-α and IL-1β.

Immunomodulatory and inflammatory effects also play a role incardiovascular conditions, such as atherogenesis and its associatedcardiovascular risks, such as atherosclerosis, thrombosis, myocardialinfarction, ischemic stroke, ischemic-reperfusion injury and peripheralvascular diseases. For example, inflammatory responses, including thoseinvolving TNF-α and IL-1β, play a role in the initiation, growth anddisruption of atheroslerotic plaques. Treatments of such cardiovascularconditions typically address hypercholesterolemia, for example, byinhibiting the enzymes involved in cholesterol biosynthesis. Statins,for example, inhibit 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase,the rate-limiting enzyme in the cholesterol biosynthesis pathway.

With heart disease being the most prevalent illness of industrializedcounties, and inflammatory conditions affecting millions of individualsworldwide, there remains a need for compounds that can treat one or bothof these types of conditions. These compounds can form the basis forpharmaceutical compositions useful in the prevention and treatment ofatherogenesis and/or inflammatory conditions in humans and othermammals. Moreover, the interplay between inflammatory and cardiovascularconditions means that compounds or combinations of compounds addressingboth may be particularly beneficial.

BRIEF SUMMARY OF THE INVENTION

In one aspect, the present invention provides compounds and compositionsthat show MAP kinase inhibitory activity and/or HMG-CoA reductaseinhibitory activity. Some embodiments are compounds comprising novelanalogs of MAP kinase inhibitors. Some embodiments are compoundscomprising novel analogs of HMG-CoA reductase inhibitors. Someembodiments are compounds comprising novel series of substitutedimidazoles, substituted pyrazoles, or substituted pyrroles. Someembodiments are componds comprising novel series of substituted indoles,substituted pyridines, substituted pyrimidines, or substitutedquinolines. Some embodiments are compounds comprising structuresmodified to favor and/or enforce a closed ring structure, e.g, aδ-lactam or a des-oxo-structure. Some embodiments are combinationscomprising two more compounds described herein and/or two or more formsof a compound described herein.

In another aspect, the present invention provides methods of treating aMAP kinase- and/or an HMG-CoA reductase-related condition byadministering an effective amount of a compound or combination ofcompounds to a subject. In some embodiments, known inhibitors of HMG-CoAreductase are used to inhibit a MAP kinase, e.g., p38α MAP kinase, inthe treatment of a MAP kinase-related condition or in the treatment ofboth a MAP kinase- and an HMG-CoA reductase-related condition. In otherembodiments, novel compounds that inhibit both a MAP kinase and HMG-CoAreductase are superior to compounds that target a MAP kinase but notHMG-CoA reductase or to compounds that target HMG-CoA reductase and notMAP kinase, for example, in treating a MAP kinase-and/or an HMG-CoAreductase-related condition. In some embodiments, novel combinations ofcompounds or forms of compounds are used to treat MAP kinase- and/orHMG-CoA reductase-related conditions that are inflammatory conditions.For example, in some embodiments, combinations comprising a statinlactone and a salt form of a hydroxy acid statin are used to treat skinand/or vascular inflammatory conditions. In preferred embodiments, suchcombinations provide synergistic effects in treating inflammation.

In another aspect, the present invention provides pharmaceuticalcompositions, formulations and modes of administering one or morecompounds, e.g., compounds of the present invention, for use in methodsof treating a MAP kinase-related and/or an HMG-CoA reeductase-relatedcondition, including inflammatory conditions. For example, in someembodiments, a statin lactone can be formulated with a hydroxy acid formof the same or different statin, a pharmaceutically acceptable saltthereof, or with another active agent. For example, in some embodiments,a statin lactone can be formulated with a non-statin anti-inflammatoryagent. Such combination formulations are administered orally ortopically in preferred embodiments, e.g., in the treatment ofinflammatory conditions.

In yet another aspect, the present invention provides methods for therational design of inhibitors of MAP kinase, HMG-CoA reductase, or bothfor use in the practice of the present invention. In certainembodiments, such methods involve designing a compound comprising alipophilic moiety, e.g., a lipophilic MAP kinase inhibitor, or a moietyor analog thereof, or comprising a lipophilic moiety or analog of anHMG-CoA reductase inhibitor; testing the designed compound for MAPkinase and/or HMG-CoA reductase inhibitory activity; and using thecompound to make a composition for inhibiting MAP kinase and/or HMG-CoAreductase, e.g., for use in the practice of the present invention. Insome embodiments, two or more designed compounds or forms thereof areused in preparing combination formulations, e.g., as described herein.

In one aspect, the present invention provides compositions that show MAPkinase inhibitory and/or 3-hydroxy-3-methyl glutaryl-coenzyme Areductase (HMG-CoA reductase) inhibitory activity. Some embodiments arecompositions comprising novel analogs of MAP kinase inhibitors. Someembodiments are compositions comprising novel analogs of HMG-CoAreductase inhibitors. Some embodiments are compositions comprisingstructures modified to favor and/or enforce a closed ring structure,e.g., a δ-lactam or a des-oxo-structure.

In another aspect, the present invention provides pharmaceuticalcompositions comprising combinations of a lactone form of a “statin”inhibitor of HMG-CoA reductase with one or more additionalpharmacologically active agents. Some embodiments are compositionscomprising a statin lactone and the hydroxy acid form of a statin, or apharmaceutically acceptable salt thereof. An embodiment of the inventionincludes compositions where the ratio of lactone statin to hydroxy acidstatin is between about 99:1 and about 1:99. Preferred embodimentsinclude a composition comprising an atorvastatin lactone and acomposition comprising an atorvastatin hydroxy acid or a pitavastatinhydroxy acid. Some embodiments are compositions comprising a statinlactone and a non-statin anti-inflammatory agent. An embodiment of theinvention includes compositions where the ratio of lactone statin tonon-statin anti-inflammatory agent is between about 99:1 and about 1:99.Preferred embodiments include compositions comprising an atorvastatinlactone and/or indomethacin. In a most preferred embodiment, acomposition comprising an atorvastatin lactone and indomethacin has asynergistic effect.

In another aspect, the present invention provides methods of treating aninflammatory condition by administering an effective amount of apharmaceutical composition, e.g., a composition of the presentinvention, to a subject. In other embodiments, the invention providesmethods which target both a MAP kinase, e.g., p38α MAP kinase andHMG-CoA reductase for inhibition that are superior to methods that thattarget a MAP kinase but not HMG-CoA reductase or to methods that targetHMG-CoA reductase and not MAP kinase, for example, in the treatment of aMAP kinase- and/or an HMG-CoA reductase-related condition.

In another aspect, the present invention relates to the use, in thetreatment of a MAP kinase-related conditions that are inflammatorydiseases and disorders associated with inflammation, of pharmaceuticalcompositions comprising combinations of a lactone form of a “statin”inhibitor of the enzyme 3-hydroxy-3-methyl glutaryl-coenzyme A reductase(HMG-CoA reductase) with one or more additional pharmacologically activeagents. Some embodiments are the use of a pharmaceutical compositioncomprising a combination of a statin lactone and a hydroxy acid statinsalt, e.g., as a therapy to treat inflammatory diseases and disordersassociated with inflammation, preferably vascular diseases anddisorders. Some embodiments are the use of a pharmaceutical compositioncomprising a combination of a statin lactone and a hydroxy acid statinsalt, e.g., as a topical therapy to treat inflammatory diseases anddisorders of the skin. Some embodiments are the use of a pharmaceuticalcomposition comprising a combination of a statin lactone and anon-statin anti-inflammatory agent, e.g., as a therapy to treatinflammatory diseases and disorders. In some embodiments, a combinationof a statin lactone and another active agent provides a synergisticeffect in treating a MAP kinase-related condition, e.g., an inflammatorycondition.

One aspect of the present invention provides methods of inhibiting a MAPkinase comprising administering an effective amount of at least onecompound comprising formula V:

-   wherein R₁ is

-   n being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

Another aspect of the present invention provides a compound comprisingformula V:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₄ is the pyridinyl ring optionally    substituted with one or more substituents selected from halogen    atoms and hydroxyl, C₁₋₃ alkyl, C₁₋₃ alkoxy and trifluoromethyl    groups, then the bridging group of R₁ is —CH₂—CH₂—.

Some embodiments provide a compound comprising formula V:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₄ is the pyridinyl ring, said pyridinyl    ring is substituted with one or more optionally substituted amino    groups.

In some embodiments, a compound comprising formula Va is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyrimidinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

In some embodiments, a compound comprising formula Vb is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyridinyl ring is optionally substituted, with the proviso that    when the pyridinyl ring is unsubstituted or substituted with one or    more substituents selected from halogen atoms and hydroxyl, C₁₋₃    alkyl, C₁₋₃ alkoxy and trifluoromethyl groups, then the bridging    group of R₁ is —CH₂—CH₂—;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

Another aspect of the present invention provides methods of inhibiting aMAP kinase comprising administering an effective amount of at least onecompound comprising formula VI:

-   wherein R₁ is

-   n being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    therof.

Another aspect of the present invention provides a compound comprisingformula VI:

-   wherein R₁ is

-   n being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,    trifluoromethyl, diakylamino where alkyl is 1–4 carbon atoms,    pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

Some embodiments provide a compound comprising formula VI:

-   wherein R₁ is

-   n being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

In some embodiments, a compound comprising formula VIa is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyrimidinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,    trifluoromethyl, diakylamino in which alkyl is 1–4 carbon atoms,    pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

In some embodiment, a compound comprising formula VIb is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyridinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof, with the proviso that when R₂ is an alkyl from 1–3 carbon    atoms, trifluoromethyl, diakylamino in which alkyl is 1–4 carbon    atoms, pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

Another aspect of the present invention provides methods of inhibiting aMAP kinase comprising administering an effective amount of at least onecompound comprising formula VII

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₃ is any substituent;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

Another aspect of the present invention provides a compound comprisingformula VII:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₃ is any substituent;-   R₄ is optionally substituted

-   optionally substituted

-   or substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

Some embodiments provide a compound comprising formula VII:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₃ is any substituent;-   R₄ is substituted

-   wherein said R₄ is substituted with one or more optionally    substituted amino groups or optionally substituted alkoxy groups;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

In some embodiments, a compound comprising formula VIIa is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyridinyl ring is substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

In some embodiments, a compound comprising formula VIb is provided:

-   wherein R₁ is

-   being 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyrimidinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

In some embodiments, the inhibited MAP kinase is p38 MAP kinase. In someembodiments, the method further comprises inhibiting an HMG CoAreductase. In some embodiments, the administering treats a MAPkinase-related condition. In some embodiments, the administering treatsa MAP kinase-related condition and an HMG CoA reductase-relatedcondition. In some embodiments, the administering treats an inflammatorycondition.

Still another aspect of the instant invention provides a pharmaceuticalcomposition comprising an effective amount of at least one compound asrecited above with a pharmaceutically acceptable carrier.

Still other aspects of the instant invention provide methods of treatinga condition in a subject in need thereof comprising administering to thesubject an effective amount of at least one compound comprising formulaV, VI and/or VII.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates some of the pathways involved in inflammatorysignaling cascades and the interruption of certain of these pathways bya MAP kinase inhibitor.

FIG. 2 a illustrates some of the pathways involved in cholesterolbiosynthesis and some of the atherogenic mechanisms ofhypercholesterolemia, as well as the interruption of certain of thesepathways by an HMG-CoA reductase inhibitor.

FIG. 2 b illustrates some of the pathways involved in processing amyloidprecursor protein, the role played by cholesterol in such pathways, aswell as the interruption of certain of these pathways by an HMG-CoAreductase inhibitor.

FIG. 3 illustrates the inter-conversion of a lactone (formula I) andacid forms (formulas IIa and IIb) of a compound of the presentinvention; FIG. 3 a illustrates non-specific configurations at each ofthe two stereogenic centers of formulas I, IIa, and IIb; FIG. 3 billustrates a preferred absolute configuration, designated (T,T).

FIG. 4 illustrates one or more examples of each of twelve classes (a–l)of inhibitors of p38α MAP kinase.

FIG. 5 illustrates lactone derivatives of one example each of twelveclasses (a–l) of inhibitors of p38α MAP kinase.

FIG. 6 illustrates an example of each of twelve classes (a–l) of statininhibitors of HMG-CoA reductase in lactone form.

FIG. 7 illustrates examples of lipophilic moieties of six classes (a–f)of synthetic statin inhibitors of HMG-CoA reductase and theirsubstituents.

FIG. 8 illustrates specific examples of lactone derivatives of each offour classes (a–d) of statin inhibitors of HMG-CoA reductase.

FIG. 9 illustrates two examples of modified closed ring structures of aδ-lactone; FIG. 9 a represents a des-oxo-form (formula III); FIG. 9 brepresents a δ-lactam form. (formula IV).

FIG. 10 illustrates a treatment approach in which compositions of thepresent invention produce a benefit in both MAP kinase-related andHMG-CoA reductase-related conditions.

FIG. 11 illustrates a design approach for developing compounds thatinhibit MAP kinase and/or HMG-CoA reductase; FIG. 11 a illustrates anapproach in which known inhibitors of MAP kinases are systematicallyvaried and tested for HMG-CoA reductase inhibitory activity; FIG. 11 billustrates an approach in which known inhibitors of HMG-CoA reductaseare systematically varied and tested for MAP kinase inhibitory activity;FIG. 11 c illustrates an approach in which the lipophilic moiety ofcompounds of formula I or II is varied and tested for MAP kinase and/orHMG-CoA reductase inhibitory activity.

DETAILED DESCRIPTION OF THE INVENTION

I. Kinase and/or HMG-CoA Reductase Inhibitors

One aspect of the present invention relates to compounds that inhibitprotein kinases, e.g., protein kinases involved in inflammatorysignaling cascades. In some embodiments, these compounds can inhibitmitogen-activated protein kinases (MAP kinases). For example, thesecompounds can inhibit p38 MAP kinases and/or stress-activated proteinkinases/Jun N-terminal kinases (SAPKs/JNKs). In some embodiments, thesecompounds can inhibit p38α MAP kinase. In preferred embodiments, suchcompounds exert anti-inflammatory effects in vitro and in vivo, e.g., asdescribed in more detail below.

FIG. 1 illustrates some of the pathways involved in inflammatorysignaling cascades and the interruption of certain of these pathways bya MAP kinase inhibitor. This figure provides an overview only, and is inno way intended to be limiting with respect to the present invention.For example, those skilled in the art will readily appreciate variationsand modifications of the scheme illustrated.

As FIG. 1 illustrates, inflammatory signaling cascades transmit signalsfrom outside a cell membrane 101 to the cytoplasm 102 and ultimately thenucleus 103. Pro-inflammatory cytokines 104 (e.g., TNF-α and IL-1), aswell as cellular stresses 105 and growth factors 106, initiate a signaltransduction cascade leading to the activation of severalserine/threonine kinases, including MKK3, MKK6 and p38 MAP kinase.Chakravarty et al, Annual Reports in Medicinal Chemistry, Chapter 18,Elsevier Science (2002). As is known in the art, p38 MAP kinases existin at least four isoforms, p38α (expressed in all tissues), p38β(expressed in all tissues), p38γ (primarily expressed in skeletaltissue), and p38δ (primarily expressed in the lungs, kidneys, testes,pancreas and small intestine). One or more of these MAP kinases can beinhibited by a compound of the instant invention, or a combinationcomprising one or more such compounds, e.g., by interaction with theirshared Thr-Gly-Tyr dual phosphorylation activation motif and/or withtheir highly conserved amino acid sequences, e.g., the conserved bindingpocket for ATP. In preferred embodiments, p38α MAP kinase is inhibited,p38α MAP kinase serving as the primary MAP kinase associated with thepro-inflammatory cytokines. As such, p38α MAP kinase presents a targetfor small molecule therapeutics aimed at reducing cytokine productionand treating associated inflammatory and/or autoimmune conditions.

As FIG. 1 illustrates, activation of p38 MAP kinase by upstream kinasesleads to phosphorylation of downstream substrates, including MNK andMAPKAP-2, as well as transcription factors ATF-2, Elk-1, and MSK-1,which control transcription and production of pro-inflammatorycytokines. FIG. 1 also illustrates points of action of an inhibitor thatcan reduce downstream effects of p38 MAP kinase, illustrated by doublebars. For example, inhibition of p38α MAP kinase using a compound of thepresent invention, or a composition comprising one or more suchcompounds, can reduce phosphorylation of MNK, MAPKAP-2, ATF-2, Elk-1and/or MSK-1, reducing production of pro-inflammatory cytokines, incertain embodiments, as discussed in detail below.

A second aspect of the present invention relates to compounds that caninhibit the enzyme 3-hydroxy-3-methyl glutaryl-coenzyme A reductase(HMG-CoA reductase). These compounds can lower cholesterol levels invitro and in vivo. FIG. 2 a illustrates some of the pathways involved incholesterol biosynthesis and some of the atherogenic mechanisms ofhypercholesteremia, as well as the interruption of certain of thesepathways by an HMG-CoA reductase inhibitor. This figure provides anoverview only, and is in no way intended to be limiting. For example,those skilled in the art will readily appreciate variations andmodifications of the scheme illustrated, and more detailed descriptionscan be found in standard texts on biochemistry, metabolism,pathophysiology, and the like.

As is known in the art, HMG-CoA reductase catalyzes the committed,rate-limiting step of terpene and cholesterol synthesis in mammaliancells. It thus represents a target for small molecule therapeutics(e.g., the “statins”) aimed at reducing atherogenesis and its associatedcardiovascular risks. HMG-CoA reductase acts on3-hydroxy-3-methyl-glutaryl CoA (HMG-CoA) to produce mevalonate.Mevalonate is converted into cholesterol, which is carried in the bloodmainly in two specialized particles known as low-density lipoprotein(LDL) and high-density lipoprotein (HDL). The pathway also producesother non-sterol isoprenoid products, such as famesol, dolichol, andubiquinone.

As illustrated in FIG. 2 a, LDL adheres to the arterial wall and isprogressively oxidized. Palinski et al., J. Am. Soc. Nephrol., 13:1673–1681 (2002). Extensively oxidized LDL is taken up by macrophages toform foam cells, a key feature of atherosclerosis. This leads torecruitment of monocytes and T-cells and secretion of cytokines inimmune response cascades. The double bars indicate currently knowneffects of HMG-CoA reductase inhibitors (e.g., statins) on theseprocesses, not only in reducing the production of cholesterol, but alsoin modulating immune responses through the actions of other metabolitessuch as farnesyl pyrophosphate and geranylgeranyl pyrophosphate. Forexample, geranylgeranyl-PP decreases endothelial cell nitric oxidesynthase (eNOS) expression, inhibiting nitric oxide-inducedvasodilation. Inhibition of HMG CoA reductase using a compound of thepresent invention, or a composition comprising one or more suchcompounds, can also produce these effects, in certain embodiments, asdiscussed in detail below.

A compound of the present invention, or a composition comprising one ormore such compounds, can increase HDL levels (“good cholesterol”) insome embodiments. HDL plays a role in carrying excess cellularcholesterol in what is known as the reverse cholesterol transportpathway. Generally, HDL is a complex of protein, lipids and cholesterol,which “scours” the walls of blood vessels to remove excess cholesterol.In reverse cholesterol transport, peripheral tissues (e.g., vessel-wallmacrophages) remove excess cholesterol through ABCA1 to apolipoproteinA-I, forming pre-β-HDL. Lecithin-cholesterol acyltransferase thenesterifies free cholesterol to cholesteryl esters, converting pre-β-HDLto mature spherical A-HDL. Forrester, J. S., Makkar, R., Shah, P. K.Circulation 111: 1847–1854 (2005), incorporated herein by reference. Acompound of the present invention, or a combination comprising one ormore such compounds, can decrease serum LDL/HDL ratios, in someembodiments.

Several steps in the cholesterol biosynthesis pathway have beenimplicated in Alzheimer's disease-related processes. Alzheimer's hasbeen linked to several proteins of the cholesterol biosynthesis pathway.As is known in the art, neuronal cells obtain cholesterol in two ways:through de novo synthesis or by internalizatioin through endosomalmechanisms. Cells which utilize the former synthesize cholesterol denovo in the endoplasmic reticulum and thereafter transport it to thecell membrane. Cells that utilize the latter internalize cholesterolsynthesized by other neuronal cells such as astrocytes. For example,cholesterol secreted via the ATP-binding cassette transporter 1 (ABCA1)transporter protein is taken up by brain HDL, containing apoliproteins Eand J. Cholesterol-containing brain HDL can be internalized by neuronalcells through an extracellular membrane receptor, called low-densitylipoprotein-related receptor (LRP). Uptake is further assisted by LRP8and very-low-density lipoprotein receptor (VLDLR). Polymorphisms ingenes encoding cholesterol pathway proteins are putative risk factorsfor Alzheimer's. Such cholesterol pathway proteins include, e.g., thetransport molecule apolipoprotein E, the uptake molecules LRP, LRP8, andVLDLR, as well as ABCA1 (a catabolism-related molecule), and Cyp46 (anoxysterol producer). Wolozin, W., Cholesterol, statins and dementia(review), Curr. Op. Lipidol. 15:667–672 (2004).

The pathology of Alzheimer's disease is characterized by the presence ofneuritic plaques composed largely of β-amyloid (Aβ) protein fragments.Aβ is produced when membrane bound amyloid precursor protein (APP) iscleaved by proteolytic enzymes, β-secretase and γ-secretase. Soluble Aβfragments cluster with one another to form oligomers, then fibrillar Aβaggregates, and eventually neuritic Aβ plaques.

FIG. 2 b illustrates some of the pathways involved in processing amyloidprecursor protein, the role played by cholesterol in such pathways, aswell as the interruption of certain of these pathways by an HMG-CoAreductase inhibitor. This figure provides an overview only, and is in noway intended to be limiting. For example, those skilled in the art willreadily appreciate variations and modifications of the schemeillustrated, and more detailed descriptions can be found in standardtexts on biochemistry, metabolism, pathophysiology, and the like.

As shown in FIG. 2 b, a cholesterol-rich membrane is required forproteolysis of APP, which subsequently leads to the production of Aβ andeventual Aβ plaque formation. Wolozin, W., Cholesterol, statins anddementia (review), Curr. Op. Lipidol. 15:667–672 (2004). Cell 1 of FIG.2 b shows a neuronal cell in the process of synthesizing its owncholesterol de novo and then transporting it to the cell membrane toallow APP processing. Cell 2 shows a neuronal cell in the process ofsynthesizing and secreting it through the ABCA1 transporter proteinwhere brain HDL protein binds cholesterol. Cell 3 shows a neuronal cellin the process of internalizing the HDL-cholesterol complex by way ofLRP. The subsequent transport of cholesterol to Cell 3's membrane allowsAPP processing to occur. FIG. 2 b also illustrates how inhibition of HMGCoA reductase in Cell 1 and Cell 2 using an HMG CoA reductase inhibitorcan produce an inhibitory effect on cholesterol synthesis and therebyaffect APP processing. The double bars indicate currently known effectsof HMG-CoA reductase inhibitors (e.g., statins) on these processes. Forexample, HMG-CoA reductase inhibitors have been found to reduceβ-secretase proteolysis of APP in cultured human cells overexpressingAPP, while applying solubilized cholesterol to such cells resulted in asignificant increase in Aβ products. In addition, reducing cellularcholesterol levels in hippocampal neurons has been shown to inhibit Aβformation. Reiss, A. B. et al., Cholesterol in neurologic disorders ofthe elderly: stroke and Alzheimer's disease (review), Neurobiology ofAging 25:977–89 (2004). Inhibition of HMG CoA reductase using a compoundof the present invention, or a composition comprising one or more suchcompounds, can also produce these effects, in certain embodiments, asdiscussed in detail below.

A third aspect of this invention relates to compounds that inhibit bothMAP kinase and HMG-CoA reductase activities. Such compounds can inhibitboth inflammatory responses and cholesterol biosynthetic pathways invitro and in vivo, and can exert, for example, anti-inflammatory,lipid-modulating, and anti-atherogenic properties in vivo. Further, suchcompounds can provide superior benefits in treating HMG-CoAreductase-related conditions, such as cardiovascular disease, comparedwith treatments that inhibit HMG-CoA reductase but not MAP kinase, dueto the interplay between inflammatory and cardiovascular disorders. Inother embodiments, such compounds can provide superior benefits intreating MAP kinase-related conditions, such as inflammation, comparedwith treatments that inhibit MAP kinase but not HMG-CoA reductase, againdue to the interplay between inflammatory and cardiovascular conditions.

A fourth aspect of this invention relates to combinations of two or morecompounds or forms of compounds that inhibit MAP kinase and/or HMG-CoAreductase activities, e.g., to produce one or more of the effectsdescribed above. Such combinations find particular use in treatinginflammatory conditions. Without being limited to a particularhypothesis, theory or mechanism, HMG-CoA reductase and MAP kinase mayboth play a role in certain inflammatory conditions, making the use ofcombination therapies particularly effective. For example, HMG-CoAreductase and MAP kinase both have been implicated in inflammatoryconditions of the skin.

Acne is an example of a skin inflammatory conditon involving activitiesof both MAP kinase and HMG-CoA reductase. Acne results from theformation of a comedone followed by pericomedonian inflammation (orfolliculitis). A comedone (or blackhead) forms when a pilo-sebaceousduct is obstructed and/or when there is increased production of sebum bya sebaceous gland. Formation of the comedone is followed byinflammation, e.g., resulting from bacterial proliferation due toseborrhoeic retention and/or overproduction of sebum. Typically, thebacteria are diphtheroid anaerobic bacteria such as Propionibacteria(acnes, granulosum, avidum). In addition to inflammatory pathways,pathways involving HMG-CoA reductase may also be involved. For example,it is known in the art that cholesterol and the metabolites thereof playa role in cohesion of epidermal cells, particularly comeocytes (cellsconstituting the stratum corneum).

As another example, psoriasis is a chronic hyperproliferative skincondition wherein the subject exhibits inflammation, as well as excessproliferation of epidermal cells (scaling). The cause is thought to bean abnormal immune response to some element of the skin prompted bymalfunctioning T cells. It is known in the art that multiple cellularevents occur at the response site including increased cell adhesionmolecule expression, upregulation of cytokines and growth factors, andpenetration of the tissue by lymphocytes. It is also known in the artthat HMG-CoA reductase inhibitors downregulate expression of celladhesion molecules, inhibit the interaction between adhesion moleculesrequired for leukocyte infiltration into inflammation sites, suppressthe expression of T-helper-1 chemokine receptors on T cells, and inhibitthe expression of proinflammatory cytokines. Namazi, M. R., ExperimentalDermatology, 13:337–39 (2004). As another example, is known in the artthat a form of eczema, atopic dermatitis, is mediated by theinflammatory mediators IFN-γ and/or TNF-α.

HMG-CoA reductase and/or MAP kinase may also play a role inmuscoskeletal inflammatory conditions, such as arthritis, psoriaticarthritis, osteoarthritis, rheumatoid arthritis, and osteoporosis. Forexample, pathological bone resorption or erosion in osteoporosis andrheumatoid arthritis requires the activation of osteoclasts (largemultinucleate cells formed from differentiated macrophages) and TNF-α,IFN-γ and IL-1 have been implicated in triggering excess osteoclastactivity. Roux, S. Bone loss. Factors that regulate osteoclastdifferentiation: an update (review), Arthritis Res., 2(6):451–456(2000); Evans et al., Nitric oxide and bone (review), J Bone Miner Res.March;11(3):300–5 (1996).

HMG-CoA reductase and/or MAP kinase may also play a role in respiratoryinflammatory conditions. For example, the inflammatory mediators IFN-γand/or TNF-α are known in the art to mediate asthma and mucocutaneousinflammatory conditions such as allergic rhinitis.

HMG-CoA reductase and/or MAP kinase may also play a role ingastrointestinal and urinogenital inflammatory conditions. For example,gastrointestinal inflammatory conditons, such as inflammatory boweldisease (including ulcerative colitis and Crohn's disease), celiacdisease, intestinal infections, enterocolitis, and gastritis, exhibitchronic spontaneous relapsing enteropathies mediated by IFN-γ and TNF-αFurther, it is known in the art that urogenital inflammatory disordersare mediated by IFN-γ and TNF-α.

HMG-CoA reductase and/or MAP kinase may also play a role in autoimmunediseases. For example, according to recent reports, HMG-CoA reductaseinhibitors may have a beneficial effect on autoimmune disorders, such asmultiple sclerosis (MS). Stuve, O., et al., The potential therapeuticrole of statins in central nervous system autoimmune disorders (review),Cell Mol Life Sci. 2003 November;60(11):2483–91. Generally, MS ismediated by proinflammatory CD4 T (Th1) cells that recognize specificmyelin proteins associated with MHC class II molecules on antigenpresenting cells (APCs). It is known in the art that inhibitors ofHMG-CoA reductase inhibit the production of iNOS, TNF-α, IL-1beta andIL-6 by microglia and astrocytes, both APCs. HMG-CoA reductaseinhibitors also inhibit IFN-γ-inducible class II expression on APCs,e.g., by inhibiting transcription of the IFN-γ-inducible promoter, whichmay result in suppression of antigen presentation by APCs. Some HMG-CoAreductase inhibitors also bind lymphocyte function-associated antigen-1(LFA-1), a beta2-integrin and prevent interaction with its ligand,ICAM-1, as well as T cell activation, suggesting a beneficial effect onMS independent of an inhibition of HMG-CoA reductase.

HMG-CoA reductase and/or MAP kinase may also play a role in graftrejection after organ or tissue transplantation. For example, HMG-CoAreductase inhibitors have been shown to significantly reduce theincidence of organ rejection, transplant vasculopathy, and naturalkiller (NK) cell cytotoxicity in recipients of heart transplants(Kobashigawa et al., Dual roles of HMG-CoA reductase inhibitors in solidorgan transplantation: lipid lowering and immunosuppression (review),Kidney Int. Suppl., December; 52:S112–5 (1995)) and kidney transplants(Katznelson, S. et al., The effect of pravastatin on acute rejectionafter kidney transplantation—a pilot study (review), Transplantation,May 27;61(10):1469–74 (1997)). Additionally, such inhibitors have beenshown to decrease the progression of transplant vasculopathy and toincrease patient survival (Wenke, K. et al., Simvastatin reduces graftvessel disease and mortality after heart transplantation: a four-yearrandomized trial, Circulation, September 2;96(5):1398–402. (1997)),suggesting a possible drug class effect. It is known in the art thattreatment of heart and kidney transplant patients with HMG-CoA reductaseinhibitors significantly inhibits NK cell cytotoxicity beyond thatobtained with the baseline regimen, consisting of prednisone,azathioprine, and cyclosporine. For example, it is known in the art thatclinically relevant concentrations of simvastatin, which are notimmunosuppressive themselves, significantly enhance inhibition of humanT-cell responses by cyclosporin A in vitro. It has been suggested thatsynergism between the inhibitors and cyclosporin A could potentially bethe basis for the immunosuppression uniquely observed in transplantpatients. Katznelson, S. et al., Effect of HMG-CoA reductase inhibitorson chronic allograft rejection (Review), Kidney Int Suppl. 1999July;71:S117–21 (1999).

Accordingly, inhibition of HMG CoA reductase and/or MAP kinase,preferably inhibition of both, by a combination of compounds or forms ofcompounds of the present invention can also produce the aforementionedeffects, in certain embodiments, as discussed in detail below.

In certain embodiments, the compositions of the present inventioncomprise compounds of formulas I and/or II, wherein I is a δ-lactone(cyclic ester) and II is a 3,5-dihydroxy carboxylic acid in protonatedform (formula IIa) or deprotonated form (formula IIb).

FIG. 3 illustrates the inter-conversion between δ-lactone and acidforms, where the δ-lactone ring opens to the acid form with (reversible)addition of water, and further equilibriates to the deprotonated formwith the loss of a proton to give the corresponding carboxylate ion. Itwill be recognized by those in the art that a rapid equilibrium existsbetween the protonated form of a carboxylic acid and its deprotonatedcarboxylate form, and that the deprotonated form predominates at neutraland basic pH. The deprotonated form is equivalent to a salt form of theacid. Further, reference to “formula II” or “II” herein refers to bothformula IIa and formula IIb, to the same extent as if the phrase“formula IIa and formula IIb” were used in place of “formula II” or“II.” Similarly, a figure or structure illustrating either the IIa orIIb form also includes the corresponding other IIb or IIa form, to thesame extent as if both structures had been illustrated. Moreover, thepresent invention encompasses both the protonated and deprotonated(i.e., salt) forms of the compounds disclosed herein.

In these formulas, X preferably comprises a lipophilic moiety. As usedherein, a lipophilic moiety can refer to a molecular entity or a portionthereof having a tendency to dissolve in fat-like solvents, e.g., in ahydrocarbon solvent. Such moieties can also be referred to ashydrophobic moieties. Preferably, X comprises a lipophilic moietybearing at least one aromatic substituent. A represents a covalent bondor a substituted or unsubstituted alkylene, alkenylene, or alkynylenelinker of 2–6 carbons, optionally containing a heteroatom, such as O, N,or S. A is preferably a covalent bond, methylene, 1,2-oxamethylene,1,2-ethylene, 1,2-ethynylene, 1,2-ethenylene, 1,3-propylene or1,3-propenylene. More preferably, A is 1,2-ethylene or E-1,2-ethenylene.Y is hydrogen or a lower alkyl, preferably hydrogen. Z is a hydroxy(—OH) group or hydrogen, preferably a hydroxy group.

In FIG. 3 a, the configurations at each of two stereogenic centers offormulas I and II are not specified. The present invention includes eachof the four possible stereoisomers arising from the two possibleabsolute configurations at each of the two stereogenic centers offormulas I and II. Compounds useful in this invention can includemixtures of the various stereoisomers or a pure stereoisomeric form.

FIG. 3 b illustrates an absolute configuration, designated (T,T),preferred in some embodiments of this invention. The designation (T,T)as used herein refers to the stereochemistry indicated, whereinwedge-shaped solid lines indicate bonds protruding above the plane ofthe illustration and dashed lines indicate bonds extending below theplane, so that the Y groups are above the plane, as are the ring oxygenof formula I and the corresponding hydroxy of formula II; while the Zgroups are below the plane of the illustration. This (T,T) designationis used, rather than the conventional R or S designations, to reflectthe fact that the actual absolute stereochemistry assignment will dependon the identity of A at one of the stereogenic centers, and the identityof Y and Z at the other stereogenic center. For example, the 5-positionof the 3,5-dihydroxy carboxylic acid becomes R if A is ethylene, whereasthe stereochemistry becomes S if A is ethenylene. Based on standardrules of nomenclature and priority of substituents at stereogeniccenters, those of skill in the art can readily determine whether thestereochemistry is R or S at each of the sterogenic centers for each A,Y, and Z in the spatial arrangement depicted in FIG. 3 b. In someembodiments, the (T,T) stereoisomer may comprise more than about 50%,more than about 70%, preferably more than about 90%, and more preferablymore than about 98% of a mixture of more than one stereoisomer.

Further, those of skill in the art will recognize that certain compoundsof the present invention may exhibit the phenomena of tautomerism,conformational isomerism, geometric isomerism and/or optical isomerism.It should be understood that the invention encompasses any tautomeric,conformational isomeric, optical isomeric and/or geometric isomericforms of the MAP kinase and/or HMG-CoA reductase inhibitors describedherein, as well as mixtures of these various different forms. Forexample, optically active compounds of the present invention may beadministered in enantiomerically pure (or substantially pure) form or asa mixture of detrorotatory and levorotatory enantiomers, such as in aracemic mixture. It will also be appreciated that compounds disclosedherein can exist in different crystalline forms, including, e.g.,polymorphs. The invention encompasses these different crystalline forms,mixtures of different crystalline forms, and pure or substantially purecrystalline forms.

A. Analogs of MAP Kinase Inhibitors

A subset of the compounds of formulas I and II are novel analogs ofknown inhibitors of MAP kinases, wherein X comprises a lipophilic MAPkinase inhibitor or a lipophilic moiety of a MAP kinase inhibitor. FIG.4 illustrates one or more non-limiting examples of each of twelveclasses (a–l) of inhibitors of p38α MAP kinase.

In some embodiments, preferred analogs include those derived frompyrazoles, such as compounds 1000–1004, illustrated in FIG. 4 a. In someembodiments, preferred analogs include those derived form oxazoles, suchas compound 1005, illustrated in FIG. 4 b. In some embodiments,preferred analogs include derivatives of imidazoles, such as compounds1006–1017, as illustrated in FIG. 4 c.

In some embodiments, preferred analogs include derivatives ofpyrrolo[2,3-b]pyrimidines, such as compounds 1018 and 1019, illustratedin FIG. 4 d. In some embodiments, preferred analogs include thosederived from diazaisoquinolinones, such as compound 1020, which isillustrated in FIG. 4 e. In some embodiments, preferred analogs includethose derived from 1,2-pyrazines, such as compound 1021, illustrated inFIG. 4 f. In some embodiments, preferred analogs include derivatives ofpyrroles, such as compound 1022, illustrated in FIG. 4 g. In someembodiments, preferred analogs include derivatives of4-aminobenzophenones, such as compound 1023, illustrated in FIG. 4 h. Insome embodiments, preferred analogs include those derived from3-amidobenzamides, such as compound 1024, illustrated in FIG. 4 i. Insome embodiments, preferred analogs include those derived frompyridines, such as compound 1025, illustrated in FIG. 4 j. In someembodiments, preferred analogs include those derived frompyrimidino[4,5-d]pyrimidinones, such as compound 1026, illustrated inFIG. 4 k. In some embodiments, preferred analogs include those derivedfrom indoles, such as compound 1027, illustrated in FIG. 41.

The present invention includes all stereoisomers arising from thepossible absolute configurations at any stereogenic center of novelanalogs of MAP kinase inhibitors of formula I and/or II, e.g., wherein Xcomprises a lipophilic MAP kinase inhibitor or a lipophilic moiety of aMAP kinase inhibitor. Mixtures of the various stereoisomers or pure orsubstantially pure stereoisomeric forms may be used in variousembodiments of the instant invention.

In certain embodiments, lipophilic MAP kinase inhibitors are substitutedor appended with A-lactone or A-acid moieties of formulas I and II,respectively, to form novel compounds of the present invention. FIG. 5illustrates lactone (formula I) derivatives of one example of each ofthe twelve classes of inhibitors of p38α MAP kinase illustrated in FIG.4. It is to be understood that these represent examples of certainembodiments, and that other kinase inhibitors, attachment points,linking moieties (A), stereochemistries, etc., are expresslycontemplated and included as other embodiments of the present invention.Further, the acid forms (formula II) of each and any of these examplesare also a contemplated embodiment of the present invention.

FIG. 5 a illustrates some preferred compounds of formula I wherein Xcomprises the pyrazole MAP kinase inhibitor compound 1000 or alipophilic moiety thereof substituted or appended at three differentpositions with the A-lactone moiety, and wherein A is 1,2-ethenylene or1,2-ethylene and Y is hydrogen.

FIG. 5 b illustrates some preferred compounds of formula I wherein Xcomprises a lipophilic moiety of the oxazole MAP kinase inhibitorcompound 1005, and wherein A is 1,2-ethenylene and Y is hydrogen. Ineach of the two structures illustrated, one of compound 1005'sappendages on the oxazole ring has been substituted by the A-lactonemoiety of formula I. The two structures illustrated in FIG. 5 b arepreferred compounds in some embodiments.

FIG. 5 c illustrates some preferred compounds of formula I wherein Xcomprises the imidazole MAP kinase inhibitor compound 1006 or alipophilic moiety thereof, A is 1,2-ethenylene and Y is hydrogen. In oneof the three illustrated examples, the lipophilic moiety comprises allof compound 1006 except the 4-piperidyl moiety, which has been replacedwith the A-lactone moiety of formula I. In another of the illustratedexamples, the lipophilic moiety comprises all of compound 1006 exceptthe 2-methoxy-4-pyrimidinyl group, which has been replaced. The threestructures illustrated in FIG. 5 c are preferred compounds in someembodiments of the present invention.

FIG. 5 d illustrates some preferred compounds of formula I wherein Xcomprises a lipophilic moiety of pyrrolo[2,3-b]pyrimidine MAP kinaseinhibitor compound 1018, A is 1,2-ethenylene, and Y is hydrogen. In theillustrated examples, the lipophilic moieties each comprise all but oneof the aromatic substituents of compound 1018 in that the A-lactonemoiety of formula I has been substituted for one of these substituents.The two structures illustrated in FIG. 5 d are preferred compounds insome embodiments.

FIG. 5 e illustrates some preferred compounds of formula I wherein Xcomprises the diazaisoquinolinone MAP kinase inhibitor compound 1020 ora lipophilic moiety thereof, A is 1,2-ethenylene or 1,2-methenomethyleneand Y is hydrogen. In two of the illustrated examples, the lipophilicmoiety comprises all but a thioaryl substituent, or all but one carbonyloxygen substituents, of compound 1020 in that the A-lactone moiety offormula I has been substituted for one of each of these substituents.

FIG. 5 f illustrates some preferred compounds of formula I wherein Xcomprises the 1,2-pyrazine MAP kinase inhibitor compound 1021 or alipohilic moiety thereof, A is 1,2-ethenylene and Y is hydrogen. In theillustrated examples, the lipophilic moiety comprises all but onearomatic substituent, or all but a benzene ring, or all but a piperazinering of compound 1021 in that the A-lactone moiety of formula I has beensubstituted for one of each of these substituents.

FIG. 5 g illustrates some preferred compounds of formula I wherein Xcomprises the pyrrole MAP kinase inhibitor compound 1022 or a lipophilicmoiety thereof, A is 1,2-ethylene or 1,2-ethenylene, and Y is hydrogen.In one of the illustrated examples, the lipophilic moiety comprises allbut a carboxymethyl group of compound 1022 in that the A-lactone moietyof formula I has been substituted for this substituent. The threestructures illustrated in FIG. 5 g represent preferred compounds in someembodiments.

FIG. 5 h illustrates some preferred compounds of formula I wherein Xcomprises the 4-aminobenzophenone MAP kinase inhibitor compound 1023, Ais 1,2-ethenylene, and Y is hydrogen. In the illustrated examples, thecompound 1023 structure is appended at three different positions withthe A-lactone moiety of formula I. The three structures illustrated inFIG. 5 h represent preferred compounds in some embodiments.

FIG. 5 i illustrates some preferred compounds of formula I wherein Xcomprises the 3-amidobenzamide MAP kinase inhibitor compound 1024, A is1,2-ethenylene, and Y is hydrogen. In the illustrated examples, thecompound 1024 structure is appended at three different positions withthe A-lactone moiety of formula I.

FIG. 5 j illustrates some preferred compounds of formula I wherein Xcomprises the pyridine MAP kinase inhibitor compound 1025 or alipophilic moiety thereof, A is 1,2-ethylene or 1,2-ethenylene, and Y ishydrogen. In one of the illustrated examples, the lipophilic moietycomprises all but a hydroxymethyl group of compound 1025 in that theA-lactone moiety of formula I has been substituted for this substituent.The first and fourth structures illustrated in FIG. 5 j are preferredcompounds in some embodiments.

FIG. 5 k illustrates some preferred compounds of formula I wherein Xcomprises the pyrimidino[4,5-d]pyrimidinone MAP kinase inhibitorcompound 1026 or a lipophilic moiety thereof, A is 1,2-methenomethylene,methylene, or 1,2-ethylene, and Y is hydrogen. In one of the illustratedexamples, the lipophilic moiety comprises all but the 4-hydroxycylohexylmoiety of compound 1026 in that the A-lactone moiety of formula I hasbeen substituted for this substituent.

FIG. 51 illustrates some preferred compounds of formula I wherein Xcomprises the indole MAP kinase inhibitor compound 1027, A is methyleneor 1,2-ethylene, and Y is hydrogen. In the illustrated examples, thecompound 1027 structure is appended at three different positions withthe A-lactone moiety. The second structure illustrated in FIG. 51 ispreferred in some embodiments.

The compounds disclosed in this invention can be produced by methodsknown in the art as they are derivatives of classes of compounds knownin the art.

The present invention relates to these compounds, to pharmaceuticalformulations comprising one of more of these compounds, e.g., incombination formulations, and to the use of such compounds and/or thecorresponding acids of formula II in treating MAP kinase-related and/orHMG-CoA reductase-related conditions, as described in more detail below.

B. Analogs of HMG-CoA Reductase Inhibitors

A subset of the compounds of formulas I and II are novel analogs ofknown inhibitors of HMG-CoA reductase, wherein X comprises a lipophilicHMG-CoA reductase inhibitor, e.g., a statin, or a lipophilic moiety ofan HMG-CoA reductase inhibitor. A statin can refer to any compound thatcan inhibit HMG-CoA reductase, generally comprising formula I or II.Known lipophilic inhibitors of HMG-CoA reductase include, for example,mevasatin, lovastatin, simvastatin, pravastatin, fluvastatin,atorvastatin, cerivastatin, rosuvastatin, pitavastatin, glenvastatin,bervastatin, dalvastatin, eptastatin, dihydroeptastatin, itavastatin,L-154819, advicor, L-654969, and other statin drugs used to treatdisorders such as hypercholesterolemia.

Statins are classified in the art as natural or synthetic statinsdepending on their origin. Natural stains include, for example,mevastatin, lovastatin, simvastatin, pravastatin, and the like.Synthetic statins include, for example, atorvastatin, fluvastatin,cerivastatin, rosuvastatin, pitavastatin, and glenvastatin.

FIG. 6 illustrates an example of each of twelve known classes (a–l) ofstatin inhibitors of HMG-CoA reductase in the lactone form of formula I.FIG. 6 a illustrates fluvastatin lactone, derivatives of which arepreferred in certain embodiments of the invention. FIG. 6 b illustratesatorvastatin lactone, derivatives of which are preferred in certainembodiments of the invention. FIG. 6 c illustrates pitavastatin lactone,derivatives of which are preferred in certain preferred embodiments.FIG. 6 d illustrates cerivastatin lactone, derivatives of which arepreferred in certain embodiments of the invention. FIG. 6 e illustratesrosuvastatin lactone, derivatives of which are preferred in certainembodiments of the invention. FIG. 6 f illustrates glenvastatin lactone,derivatives of which are preferred in certain embodiments of theinvention. FIG. 6 g illustrates simvastatin lactone, derivatives ofwhich are preferred in certain embodiments of the invention. FIG. 6 hillustrates lovastatin lactone, derivatives of which are preferred incertain embodiments of the invention. FIG. 6 i illustrates pravastatinlactone, derivatives of which are preferred in certain embodiments ofthe invention. FIG. 6 j illustrates mevastatin lactone, derivatives ofwhich are preferred in certain embodiments of the invention. FIG. 6 killustrates bervastatin lactone, derivatives of which are preferred incertain embodiments of the invention. FIG. 61 illustrates dalvastatinlactone, derivatives of which are preferred in certain embodiments ofthe invention. In particular, more preferred statin lactones are thosederived from synthetic statins, and even more preferred stain lactonesare those derived from atorvastatin, fluvastatin, rosuvastatin,cerivastatin, pitavastatin and glenvastatin.

FIG. 7, for example, illustrates more specific examples of lipophilicmoieties (X) derived from six classes (a–f) of synthetic statins andtheir substituents. In each case independently, P₁ is tert-butyl,iso-propyl, cyclopropyl, 2-hydroxy-2-propyl, 1-hydroxycyclopropyl; P₂ ishydrogen, fluorine, or -trifluoromethyl; P₃ is hydrogen, cyano, methoxy,phenoxy, anilino, phenylmethylamino, or amino; P₄ is hydrogen, phenyl orphenylcarbamoyl; P₅ is hydrogen, aryl, substituted aryl, heteroaryl,substituted heteroaryl, aniline, substituted aniline, phenyl ether,substituted phenyl ether, N-alkyl alkyl sulfonamido, N-alkyl arylsulfonamide, N-alkyl alkanamido, or N-alkyl arylamido; and Q is —CH,nitrogen, or nitrogen oxide.

FIG. 7 a illustrates an example of an indole-based lipophilic moietyrelated to fluvastatin, preferred in some embodiments. FIG. 7 billustrates two examples of imidazole-based lipophilic moieties relatedto atorvastatin, which are preferred in some embodiments. FIG. 7 cillustrates five examples of pyrrole-based lipophilic moieties relatedto atorvastatin, which are preferred in certain embodiments. FIG. 7 dillustrates three examples of quinoline-based lipophilic moietiesrelated to pitavastatin, preferred in some embodiments. FIG. 7 eillustrates four examples of pyridine-based lipophilic moieties relatedto cerivastatin, preferred in some embodiments. FIG. 7 f illustrates anexample of a pyrimidine-based lipophilic moiety related to rosuvastatin,which is preferred in some embodiments.

In certain embodiments, lipophilic derivatives of statins, including,for example the lipophilic moieties of FIG. 7, are substituted orappended with A-lactone or A-acid moieties of formulas I and II,respectively, to form novel compounds of the present invention. FIG. 8,for example, illustrates specific examples of lactone (formula I)derivatives of each of the four classes (a–d) of statins represented inFIG. 7 and wherein each of P₁, P₂, P₃, P₄, P₅, and Q have been selectedto give compounds preferred in some embodiments of the presentinvention. It is to be understood that these represent examples ofcertain embodiments, and that other statins, attachment points, linkingmoieties (A), stereochemistries, etc., are expressly contemplated andincluded as other embodiments of the present invention. Further, theacid form (formula II) of each and any of these examples is also acontemplated embodiment of the present invention.

FIG. 8 a illustrates selected indole-based lipophilic moieties relatedto fluvastatin, which are preferred in some embodiments (Family I). FIG.8 b illustrates selected imidazole-based lipophilic moieties derivedfrom atorvastatin, which are preferred in some embodiments (Family II).FIG. 8 c illustrates selected pyrrole-based lipophilic moieties derivedfrom atorvastatin, which are preferred in some embodiments (Family III).FIG. 8 d illustrates selected quinoline-based lipophilic moietiesrelated to pitavastatin, which are preferred in some embodiments,divided amongst two families, Family IV and Family V. FIG. 8 eillustrates selected pyridine-based lipophilic moieties related tocerivastatin, which are preferred in some embodiments (Family VI).

The present invention includes all stereoisomers arising from thepossible absolute configurations at any stereogenic center of novelanalogs of HMG CoA reductase inhibitors of formula I and/or II, e.g.,wherein X comprises a lipophilic HMG-CoA reductase inhibitor, e.g., astatin, or a lipophilic moiety of an HMG-CoA reductase inhibitor.Mixtures of the various stereoisomers or pure or substantially purestereoisomeric forms may be used in various embodiments of the instantinvention.

The compounds disclosed in this invention can be produced by methodsknown in the art as they are derivatives of classes of compounds knownin the art. For example, the synthesis of statins is described in Rothet al., J. Med. Chem., 34:357–366 (1991); Krause et al., J. Drug Dev.,3(Suppl. 1):255–257 (1990); and Karanewsky, et al., J. Med. Chem.33:2952–2956 (1990). Examples are also provided in the Examples below.Further, specific examples of the present invention can be made byvariations of methods known to those of skill in the art and providedherein, for example, where starting materials, solvents, and otherreaction conditions are varied to optimize yields.

In certain embodiments, the compounds of the present invention can bemade using commercially available compounds as starting materials. Forexample; lactones of formula I can be prepared from commerciallyavailable salts of HMG-CoA reductase inhibitors. For instance,commercially available calcium or sodium salts of atorvastatin,fluvastatin and rosuvastatin may be converted to their protonated freeacid forms by extracting the salt forms from weakly acidic aqueous mediainto an aprotic organic solvent such as ethyl acetate. By stirring thefree acid forms in this or another aprotic organic solvent (such astoluene) approximately at or above room temperature, spontaneousconversion to the lactone form occurs over a timeframe of about hours toabout days. The lactone forms may be conveniently purified by anymethods known in the art, including by column, preparative thin-layer,rotating, or high-pressure chromatography on silica gel columns usingstandard eluting solvent systems such as about 5:1 (v:v) acetone:ethylacetate.

In other embodiments, compounds of the present invention can be madefrom modifying intermediates of synthesis pathways of known statins. Forexample, a group can be replaced by reactive groups such as an amino,halogen, or hydroxy group, or a metal derivative such as sodium,magnesium, or lithium, and these groups further reacted. Further, thoseskilled in art will recognize that compounds of the present inventionsynthesized by various art-known methods will give cis/trans isomers,E/Z forms, diastereomers, and optical isomers, all of which are includedin the present invention.

Details for synthesizing the side chain of formula II are provided inExample 9 below.

The present invention relates to these compounds, to pharmaceuticalformulations comprising one of more of these compounds, e.g., incombination formulations, and to the use of such compounds and/or thecorresponding acids of formula II in treating MAP kinase-related and/orHMG-CoA reductase-related conditions.

Another aspect of the present invention relates to analogs of knownlipophilic MAP kinase and/or HMG-CoA reductase inhibitors, e.g. statins,having structures modified to favor and/or enforce a closed ringstructure, for example, a ring structure or cyclic form that is nothydrolyzed or not substantially hydrolyzed to its carboxylic acid orcarboxylate forms. “Not hydrolyzed” and “not substantially hydrolyzed,”along with their grammatical conjugations, include situations where someof the compound is hydrolyzed while some is not hydrolyzed. Preferably,at least about 50%, at least about 75%, at least about 90%, and morepreferably at least about 95% of the compound is in a ring structure ofcyclic form at equilibrium, in situations where the compound is notsubstantially hydrolyzed. Preferably, at least about 70%, at least about80%, at least about 90%, and more preferably at least about 95%, andeven more preferably at least about 98% of the compound is in a ringstructure or cyclic form at equilibrium, in situations where thecompound is not hydrolyzed.

FIG. 9 illustrates two examples of modified closed ring structures thatare analogs of a δ-lactone; FIG. 9 a represents a des-oxo-form (formulaIII), where the carbonyl oxygen is removed, thereby inhibitinghydrolytic ring opening. FIG. 9 b represents a δ-lactam form (formulaIV), where a nitrogen replaces an oxygen in the ring, which increasesthe hydrolytic stability of the cyclic form. In these formulas, Xcomprises a lipophilic moiety. In some preferred embodiments, Xcomprises a lipophilic MAP kinase inhibitor or a lipophilic moiety of aMAP kinase inhibitor, for example, the MAP kinase inhibitors of FIG. 4,as well as lipophilic moieties of analogs of MAP kinase inhibitors, suchas those of FIG. 5. In some preferred embodiments, X comprises alipophilic moiety of a statin, including, for example, the statins ofFIG. 6, as well as lipophilic moieties of statin analogs, such as thoseof FIGS. 7 and 8. Preferably, X comprises a lipophilic moiety bearing atleast one aromatic substituent, more preferably an aromatic moiety of asynthetic statin. A represents a covalent bond or a substituted orunsubstituted alkylene, alkenylene, or alkynylene linker of 2–6 carbons,optionally containing a heteroatom, such as O, N, or S. A is preferablya covalent bond, methylene, 1,2-oxamethylene, 1,2-ethylene,1,2-ethynylene, 1,2-ethenylene, 1,3-propylene or 1,3-propenylene. Morepreferably, A is 1,2-ethylene or E-1,2-ethenylene. Y is hydrogen or alower alkyl, preferably hydrogen. Z is a hydroxy (—OH) group orhydrogen, preferably a hydroxy group. And P₆ is hydrogen, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,substituted alkaryl, benzyl, substituted benzyl, napthylmethylene, orsubstituted napthlymethylene. Preferably, P₆ is alkaryl or substitutedalkaryl; more preferably P₆ is benzyl, substituted benzyl,napthylmethylene, or substituted napthlymethylene.

Further, each of the four possible stereoisomers, arising from the twopossible absolute configurations at each of the two stereogenic centersof formulas III and IV, are contemplated embodiments of the invention.In particular, an absolute configuration as illustrated in FIG. 3 b,depicted as (T,T), is preferred in some embodiments. Also, des-oxo andδ-lactam forms derived from synthetic statins, including, for example,atorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin, andglenvastatin, are particularly preferred in some embodiments.

Some embodiments are componds comprising novel series of substitutedimidazoles, substituted pyrazoles, substituted pyrroles, substitutedindoles, substituted pyridines, substituted pyrimidines, or substituedquinolines, some embodiments of which are discussed in more detailbelow.

The present invention relates to these compounds, to pharmaceuticalformulations comprising one of more of these compounds, e.g., incombination formulations, and to the use of such compounds and/or thecorresponding acids of formula II in treating MAP kinase-related and/orHMG-CoA reductase-related conditions, as described in more detail below.

C. Substituted Imidazole Series

In some aspects of the invention, methods described herein employ asubset of the compounds of formulas I and II that are substitutedimidazoles of formula V

-   where R₁ is

-   in which n is 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

The dotted line in the bridging group of R₁ is meant to indicate thatthe bridging group may be either an ethyl (i.e., is —CH₂—CH₂—) orethenyl (i.e., —CH═CH—) group. Also contemplated as falling within thescope of formula V are salts, solvates, esters, tautomers, polymorphs,metabolites, prodrugs, N-oxides, sulfoxides or sulfones thereof.Preferred salts include those of calcium, sodium and potassium.

In some embodiments, the N at the 3-position of the imidazole ring isprotonated (e.g., reversibly protonated) or optionally substituted. Alsocontemplated within the scope of Formula V are compounds where the R₄pyrimidinyl, pyridinyl or imidazolyl ring is attached via any availablecarbon or nitrogen atom of the ring.

In choosing compounds of the present invention, one of ordinary skill inthe art will recognize that the various substituents, i.e. R₁, R₂, etc.,are to be chosen in conformity with well-known principles of chemicalstructure connectivity.

The term “substituted” can include multiple degrees of substitution by anamed substitutent. Where multiple substituent moieties are disclosed orclaimed, the substituted compound can be independently substituted withone or more of the disclosed or claimed substituent moieties, singly orpluraly.

“Alkyl”, as well as other groups having the prefix “alk”, such asalkoxy, alkanoyl, can refer to optionally substituted carbon chainswhich may be linear or branched or combinations thereof. Examples ofalkyl groups include, e.g., methyl, ethyl, n-propyl, isopropyl, n-butyl,iso- sec- and tert-butyl, pentyl, hexyl, heptyl, octyl, nonyl, and thelike.

“Aryl” can refer to optionally substituted mono- or bicyclic aromaticrings containing only carbon atoms. The term can also include aryl groupfused to a monocyclic cycloalkyl or monocyclic cycloheteroalkyl group inwhich the point of attachment is on an aromatic portion. Examples ofaryl groups include, e.g., phenyl, naphthyl, indanyl, indenyl,tetrahydronaphthyl, 2,3-dihydrobenzofuranyl, dihydrobenzopyranyl,1,4-benzodioxanyl, and the like.

“Heteroaryl” can refer to an optionally substituted mono- or bicyclicaromatic ring containing at least one heteroatom (an atom other thancarbon), such as N, O and S, with each ring containing about 5 to about6 atoms. Examples of heteroaryl groups include, e.g., pyrrolyl,isoxazolyl, isothiazolyl, pyrazolyl, pyridyl, oxazolyl, oxadiazolyl,thiadiazolyl, thiazolyl, imidazolyl, triazolyl, tetrazolyl, furanyl,triazinyl, thienyl, pyrimidyl, pyridazinyl, pyrazinyl, benzoxazolyl,benzothiazolyl, benzimidazolyl, benzofuranyl, benzothiophenyl,furo(2,3-b)pyridyl, quinolyl, indolyl, isoquinolyl, and the like.

“Halogen” can include fluorine, chlorine, bromine and iodine.

As used herein, R, R′, etc., generally refer to any non-aromatic group,including, e.g., substituted or unsubstituted alkyl groups, unlessspecifically defined otherwise. Ar, Ar′, etc., generally refer tosubstituted or unsubstituted aromatic groups, including, e.g., aryls andheteroaryls.

Compounds of formula V contain one or more asymmetric centers and canthus occur as racemates and racemic mixtures, pure or substantially pureisomeric forms, single enantiomers, diastereomeric mixtures andindividual diastereomers. The present invention is meant to comprehendall such isomeric forms of the compounds of formula V.

In some preferred embodiments, n is 0, 1, 2 or 3. In some preferredembodiments, R₁ has the following stereochemistry:

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and/or Zgeometric isomers. In some embodiments, the E geometric isomer ispreferred.

Some of the compounds described herein may exist with different pointsof attachment of hydrogen, referred to as tautomers. Such an example maybe a ketone and its enol form known as keto-enol tautomers. Theindividual tautomers as well as mixtures thereof are encompassed withincompounds of formula V.

In preferred embodiments, the compounds of formula V are MAP kinaseinhibitors and/or are used in the methods wherein an inhibition of MAPkinase is desired, e.g., in the treatment of MAP kinase-relatedconditions.

In more preferred embodiments, a novel subset of compounds (or saltsthereof) of formula V are provided wherein R₁ is

-    in which n is 0 or any integer, preferably 0, 1 or 2; and-   R₄ is optionally substituted

-   with the proviso that when R₄ is the pyridinyl ring optionally    substituted with one or more substituents selected from halogen    atoms and hydroxyl, C₁₋₃ alkyl, C₁-₃ alkoxy and trifluoromethyl    groups, then the bridging group of R₁ is —CH₂—CH₂—.

R₄ substituents may be present on one or more vacant positions of acarbon and/or heteroatom of the pyrimidinyl, pyridinyl or imidazolylrings. Examples of suitable substituents include, but are not limitedto, oxygen, fluorine, chlorine, bromine or iodine atoms or methyl,ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy,trifluoromethyl, hydroxy, or optionally substituted amino groups. Forexample, in some embodiments, R₄ is the pyridinyl ring substituted withone or more optionally substituted amino groups, and the bridging groupof R₁ is either —CH₂—CH₂— or —CH═CH—. Particularly preferred R₄substitutents include —NH-phenyl, —NH—CH₃, and NH₂ groups.

In still some embodiments, R₄ substitutents include N-oxides, forexample:

In still some embodiments, R₄ can be

A particularly preferred group of compounds of formula V are thosewherein R₂ is a C₁₋₆ alkyl group optionally substituted with one tothree halogen atoms for example a trifluoromethyl or a C₁₋₄ alkyl group,more particularly a C₃₋₄ branched alkyl group, e.g., 1-methylpropyl. Ineven more preferred embodiments, R₂ is isopropyl, t-butyl, —CF₃, phenyl,

In some embodiments within this particularly preferred group, compoundswherein R₅ is an optionally substituted phenyl group are especiallypreferred.

When R₅ is a substituted phenyl group then these preferably contain from1 to 3 substituents. Examples of suitable R₅ substituents includehalogen atoms e.g. fluorine, bromine, chlorine, methoxy, methyl, ethyl,hydroxy or trifluoromethyl groups. Preferred substituted R₅ substituentsinclude halophenyl such as 4-fluorophenyl, 4-chlorophenyl,3-chlorophenyl, 3-bromophenyl, 3,5-dibromophenyl, 3,5-dichlorophenyl,alkyl-halophenyl such as 5-chloro-2-methylphenyl,4-fluoro-2-methylphenyl, 3,5-dimethyl-4-fluorophenyl,4-chloro-3,5-dimethylphenyl and 3,5-diethyl-4-fluorophenyl, alkylphenylsuch as 4-methylphenyl, 3,5-dimethylphenyl, hydroxyphenyl, methoxyphenylor 3-trifluoromethylphenyl. Particularly preferred is 4-fluorophenyl or3-trifluoromethylphenyl.

A particularly preferred group of compounds are those wherein R₂ is aphenyl, trifluoromethyl, t-butyl or more especially an isopropyl group;R₅ is a phenyl, 3-chlorophenyl, 3-trifluoromethylphenyl, 4-chlorophenyl,4-fluorophenyl, 4-fluoro-2-methylphenyl, 3,5-diethyl-4-fluorophenyl or3,5-dimethyl-4-fluorophenyl group; and R₄ comprises a 4-pyridinyl group,a 4-pyrimidinyl group, or a 4-imidazolyl group, a2-aminophenyl-4-pyridinyl group, a 2-aminophenyl-4-pyrimidinyl group, a2-aminomethyl-4-pyridinyl group, or a 2-aminomethyl-4-pyrimidinyl group,especially 4-pyrimidinyl, 4-pyridinyl, 2-aminophenyl-4-pyrimidinyl,2-aminomethyl-4-pyrimidinyl, 2-amino-4-pyridinyl, or2-aminomethyl-4-pyridinyl. Within this particularly preferred group ofcompounds, those in which R₅ is a 4-fluorophenyl group are especiallypreferred.

A preferred group of compounds of formula V are compounds of formula Va

-   wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2;    -   R₂ is optionally substituted alkyl, aryl, or heteroaryl;    -   the pyrimidinyl ring is optionally substituted;    -   and R₅ is optionally substituted aryl or heteroaryl, or a salt        thereof.

In some embodiments, the pyrimidinyl ring is unsubstituted. In otheremobdiments, the pyrimidinyl ring is substituted, e.g., with optionallysubstituted amino groups, especially —NH—CH₃ or —NH-phenyl; or with—O—CH₃.

Another preferred group of compounds of formula V are compounds offormula Vb

-   wherein R₁ is

-    in which n is 0 or any integer, preferably 0, 1 or 2;    -   R₂ is optionally substituted alkyl, aryl, or heteroaryl;    -   the pyridinyl ring is optionally substituted, with the proviso        that when the pyridinyl ring is unsubstituted or substituted        with one or more substituents selected from halogen atoms and        hydroxyl, C1–3 alkyl, C1–3 alkoxy and trifluoromethyl groups,        then the bridging group of R₁ is —CH₂—CH₂—;    -   and R₅ is optionally substituted aryl or heteroaryl, or a salt        thereof.

In some embodiments, the pyridinyl ring is unsubstituted. In otheremobdiments, the pyridinyl ring is substituted, e.g., with optionallysubstituted amino groups, especially, —NH₂ or —NH—CH₃.

Particularly preferred examples of compounds of the present inventioninclude, but are not limited to, the following:

More particularly preferred examples of compounds of the presentinvention include, but are not limited to, the following:

Other particularly preferred examples include, but are not limited to,the following:

Compounds of the formula V may be separated into diastereoisomenc pairsof enantiomers by, for example, fractional crystallization from asuitable solvent, for example MeOH or ethyl acetate or a mixturethereof. The pair of enantiomers may be separated into individualstereoisomers by, for example the use of an optically active amine as aresolving agent or on a chiral HPLC column. Racemic mixtures can beseparated into their individual enantiomers by any of a number ofconventional methods. These include chiral chromatography,derivatization with a chiral auxiliary followed by separation bychromatography or crystallization, and fractional crystallization ofdiastereomeric salts.

Alternatively, any enantiomer of a compound of the general formula V maybe obtained by stereospecific synthesis using optically pure startingmaterials or reagents of known configuration. In preferred embodiments,compounds of formula V are administered as enantiomerically pure (orsubstantially enantiomerically pure) formulations.

Details for synthesizing imidazoles of formula V of the invention areprovided in Examples 13 and 14 below. Specifically, Example 13 providesexamples of synthesizing(3R,5R)-7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt and(3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt, via sidechain condensation. Example 14 providesexamples of synthesizing (3S,5R),(3R,5R)-6-(5-(4-fluorophenyl)-2-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyhexanoicacid, calcium salt via an N-alkylation.

D. Substituted Pyrazole Series

In some aspects of the invention, methods described herein employ asubset of the compounds of formulas I and II that are substitutedpyrazoles of formula VI

-   where R₁ is

-   in which n is 0 or any integer;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

The dotted line in the bridging group of R₁ is meant to indicate thatthe bridging group may be either an ethyl (i.e., is —CH₂—CH₂—) orethenyl (i.e., —CH═CH—) group. Also contemplated as falling within thescope of formula VI are salts, solvates, esters, tautomers, polymorphs,metabolites, prodrugs, N-oxides, sulfoxides or sulfones thereof.Preferred salts include those of calcium, sodium and potassium.

In some embodiments, the N at the 2-position of the pyrazole ring isprotonated (e.g., reversibly protonated) or optionally substituted. Alsocontemplated within the scope of Formula VI are compounds where the R₄pyrimidinyl, pyridinyl or imidazolyl ring is attached via any availablecarbon or nitrogen atom of the ring.

In choosing compounds of the present invention, one of ordinary skill inthe art will recognize that the various substituents, i.e. R₁, R₂, etc.,are to be chosen in conformity with well-known principles of chemicalstructure connectivity.

The term “substituted” can include multiple degrees of substitution by anamed substitutent. Where multiple substituent moieties are disclosed orclaimed, the substituted compound can be independently substituted withone or more of the disclosed or claimed substituent moieties, singly orpluraly.

Compounds of formula VI contain one or more asymmetric centers and canthus occur as racemates and racemic mixtures, pure or substantially pureisomeric forms, single enantiomers, diastereomeric mixtures andindividual diastereomers. The present invention is meant to comprehendall such isomeric forms of the compounds of formula VI.

In some preferred embodiments, n is 0, 1, 2 or 3. In some preferredembodiments, R₁ has the following stereochemistry:

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and/or Zgeometric isomers. In some embodiments, the E geometric isomer ispreferred.

Some of the compounds described herein may exist with different pointsof attachment of hydrogen, referred to as tautomers. Such an example maybe a ketone and its enol form known as keto-enol tautomers. Theindividual tautomers as well as mixtures thereof are encompassed withincompounds of formula VI.

In preferred embodiments, the compounds of formula VI are MAP kinaseinhibitors and/or are used in the methods wherein an inhibition of MAPkinase is desired, e.g., in the treatment of MAP kinase-relatedconditions.

In more preferred embodiments, a novel subset of compounds (or saltsthereof) of formula VI are provided wherein R₁ is

or a salt thereof in which n is 0 or any integer, preferably 0, 1 or 2;and

-   R₄ is optionally substituted

-   with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,    trifluoromethyl, diakylamino in which alkyl is 1–4 carbon atoms,    pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

R₄ substituents may be present on one or more vacant positions of acarbon and/or heteroatom of the pyrimidinyl, pyridinyl or imidazolylrings. Examples of suitable substituents include, but are not limitedto, oxygen, fluorine, chlorine, bromine or iodine atoms or methyl,ethyl, n-propyl, isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy,trifluoromethyl, hydroxy, or optionally substituted amino groups.Particularly preferred R₄ substitutents include —OCH₃ and —NH-phenylgroups.

In still some embodiments, R₄ substitutents include N-oxides, forexample:

In still some embodiments, R₄ can be

Particularly preferred R₂ groups include lower alkyl, e.g., an alkylfrom one to four carbon atoms, dimethylamino, or 1-methylpropyl, andespecially isopropyl, phenyl, or hydrogen.

In still some preferred embodiments, R₅ is phenyl which ismonosubstituted with alkyl of from one to three carbon atoms, fluorine,chlorine, or trifluoromethyl; or phenyl which is disubstituted with twogroups independently selected from alkyl of from one to three carbonatoms, fluorine, chlorine, or trifluoromethyl. Particularly preferred is4-fluorophenyl or 3-trifluoromethylphenyl.

A particularly preferred group of compounds are those wherein R₂ is ahydrogen, phenyl, or isopropyl group; R₅ is a 3-trifluoromethylphenyl or4-fluorophenyl group; and R₄ comprises a 4-pyridinyl group, a4-pyrimidinyl group, or a 4-imidazolyl group, especially2-methoxy-4-pyrimidinyl, 2-aminophenyl-4-pyrimidinyl, or2-aminophenyl-4-pyridinyl. Within this particularly preferred group ofcompounds, those in which R₅ is a 3-trifluoromethylphenyl group areespecially preferred

A preferred group of compounds of formula VI are compounds of FormulaVIa

-   wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyrimidinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,    trifluoromethyl, diakylamino in which alkyl is 1–4 carbon atoms,    pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

In some embodiments, the pyrimidinyl ring is unsubstituted. In otherembodiments, the pyrimidinyl ring is substituted, e.g., with optionallysubstituted amino groups, especially —NH-phenyl, or with optionallysubstituted alkoxy groups, especially —O—CH₃.

Another preferred group of compounds of formula VI are compounds offormula VIb

-   wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyridinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof,-   with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,    trifluoromethyl, diakylamino in which alkyl is 1–4 carbon atoms,    pyrrolidino, piperidino, morpholino or piperazino, then R₄ is    substituted.

In some embodiments, the pyridinyl ring is unsubstituted. In otheremobdiments, the pyridinyl ring is substituted, e.g., with optionallysubstituted amino groups, especially, —NH-phenyl.

Particularly preferred examples of compounds of the present inventioninclude, but are not limited to, the following:

More particularly preferred examples of compounds of the presentinvention include, but are not limited to, the following:

Other particularly preferred examples include, but are not limited to,the following:

Compounds of the formula VI may be separated into diasteroisomeric pairsof enantiomers by, for example, fractional crystallization from asuitable solvent, for example MeOH or ethyl acetate or a mixturethereof. The pair of enantiomers may be separated into individualstereoisomers by, for example the use of an optically active amine as aresolving agent or on a chiral HPLC column. Racemic mixtures can beseparated into their individual enantiomers by any of a number ofconventional methods. These include chiral chromatography,derivatization with a chiral auxiliary followed by separation bychromatography or crystallization, and fractional crystallization ofdiastereomeric salts.

Alternatively, any enantiomer of a compound of the general formula VImay be obtained by stereospecific synthesis using optically purestarting materials or reagents of known configuration. In preferredembodiments, compounds of formula VI are administered asenantiomerically pure (or substantially enantiomerically pure)formulations.

Details for synthesizing pyrazoles of formula VI of the invention areprovided in Examples 10 and 11 below. Specifically, Example 10 providesexamples of synthesizing four N-pyridyl pyrazoles and Example 11provides examples of synthesizing eight N-pyrimidinyl pyrazoles.

E. Substituted Pyrrole Series

In some aspects of the invention, methods described herein employ asubset of the compounds of formulas I and II that are substitutedpyrroles of formula VII

-   where R₁ is

-   which n is 0 or any integer;-   R₂ is optionally substituted alkyl, aryl or heteroaryl;-   R₃ is any substituent;-   R₄ is optionally substituted

-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

The dotted line in the bridging group of R₁ is meant to indicate thatthe bridging group may be either an ethyl (i.e., is —CH₂—CH₂—) orethenyl (i.e., —CH═CH—) group. Also contemplated as falling within thescope of formula VII are salts, solvates, esters, tautomers, polymorphs,metabolites, prodrugs, N-oxides, sulfoxides or sulfones thereof.Preferred salts include those of calcium, sodium and potassium.

Also contemplated within the scope of Formula VII are compounds wherethe R₄ pyrimidinyl, pyridinyl or imidazolyl ring is attached via anyavailable carbon or nitrogen atom of the ring.

In choosing compounds of the present invention, one of ordinary skill inthe art will recognize that the various substituents, i.e. R₁, R₂, etc.,are to be chosen in conformity with well-known principles of chemicalstructure connectivity.

The term “substituted” can include multiple degrees of substitution by anamed substitutent. Where multiple substituent moieties are disclosed orclaimed, the substituted compound can be independently substituted withone or more of the disclosed or claimed substituent moieties, singly orpluraly.

Compounds of formula VII contain one or more asymmetric centers and canthus occur as racemates and racemic mixtures, pure or substantially pureisomeric forms, single enantiomers, diastereomeric mixtures andindividual diastereomers. The present invention is meant to comprehendall such isomeric forms of the compounds of formula VII.

In some preferred embodiments, n is 0, 1, 2 or 3. In some preferredembodiments, R₁ has the following stereochemistry:

Some of the compounds described herein contain olefinic double bonds,and unless specified otherwise, are meant to include both E and/or Zgeometric isomers. In some embodiments, the E geometric isomer ispreferred.

Some of the compounds described herein may exist with different pointsof attachment of hydrogen, referred to as tautomers. Such an example maybe a ketone and its enol form known as keto-enol tautomers. Theindividual tautomers as well as mixtures thereof are encompassed withincompounds of formula VII.

In preferred embodiments, the compounds of the formula VII are MAPkinase inhibitors and/or are used in the methods wherein an inhibitionof MAP kinase is desired, e.g., in the treatment of MAP kinase-relatedconditions.

In more preferred embodiments, a novel subset of compounds (or saltsthereof) of formula VII are provided wherein R₁ is wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2; and-   R₄ is optionally substituted

-   optionally substituted

-   or substituted

R₄ substituents may be present on one or more vacant positions of acarbon and/or heteroatom of the pyrimidinyl, pyridinyl or imidazolylrings. Examples of suitable substituents include, but are not limitedto, oxygen, methyl, ethyl, n-propyl, isopropyl, methoxy, ethoxy,n-propoxy, isopropoxy, trifluoromethyl, hydroxy, or optionallysubstituted amino groups. For example, in some embodiments, R₄ is thepyridinyl ring substituted with one or more optionally substituted aminogroups. Particularly preferred R₄ substitutents include —OCH₃, —NH₂,—NH—CH₃, and —NH-Phenyl.

In still some embodiments, R₄ substitutents include N-oxides, forexample:

In still some embodiments, R₄ can be

A particularly preferred group of compounds of formula VII are thosewherein R₂ is a branched alkyl group such as isopropyl or isobutyl.Particularly preferred is isopropyl.

Another preferred class of compounds of formula VII are those compoundswherein R₅ is a substituted phenyl group and preferably contains from 1to 3 substituents. Examples of suitable R₅ substituents include halogenatoms e.g. fluorine, bromine, chlorine, methoxy, methyl, ethyl, hydroxy,fluorophenyl or trifluoromethyl groups. Particularly preferred is4-fluorophenyl or 3-fluoromethylphenyl.

Another preferred class of compounds of formula VII are those compoundswherein R₃ is —CONH—W, where W is optionally substituted phenyl.Examples of suitable substituents include, but are not limited to,fluorine, chlorine, bromine or iodine atoms or methyl, ethyl, n-propyl,isopropyl, methoxy, ethoxy, n-propoxy, isopropoxy, trifluoromethyl,hydroxy, or optionally substituted amino groups. Particularly preferredare compounds in which W is unsubstituted phenyl.

A particularly preferred group of compounds are those wherein R₂ isisopropyl; R₅ is 4-fluorophenyl; R₃ is —CONH-phenyl; and R₄ comprises a4-pyrimidinyl group, 2-methyl-4-pyridinyl group, a 2-amino-4-pyridinylgroup, a 2-aminophenyl-4-pyridinyl group, a 2-aminomethyl-4-pyrimidinylgroup, a 2-aminomethyl-4-pyridinyl group, or a 4-imidazolyl group,especially 4-primidinyl, 2-methoxy-4-pyrimidinyl, 2-amino-4-pyrimidinyl,or 2-aminophenyl-4-pyrimidinyl.

A preferred group of compounds of formula VII are compounds of formulaVIIa

-   wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyrimidinyl ring is optionally substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.-   In some embodiments, the pyrimidinyl ring is unsubstituted. In other    emobdiments, the pyrimidinyl ring is substituted, e.g., with    optionally substituted amino groups, especially —NH₂ or —NH-phenyl,    or with optionally substituted alkoxy groups, especially —O—CH₃.

Another preferred group of compounds of formula VI are compounds offormula VIIb

-   wherein R₁ is

-   which n is 0 or any integer, preferably 0, 1 or 2;-   R₂ is optionally substituted alkyl, aryl, or heteroaryl;-   the pyridine ring is substituted;-   and R₅ is optionally substituted aryl or heteroaryl, or a salt    thereof.

Particularly preferred examples of compounds of the present inventioninclude, but are not limited to, the following:

More particularly preferred examples of compounds of the presentinvention include, but are not limited to, the following:

Compounds of the formula VII may be separated into diastereoisomericpairs of enantiomers by, for example, fractional crystallization from asuitable solvent, for example MeOH or ethyl acetate or a mixturethereof. The pair of enantiomers may be separated into individualstereoisomers by, for example the use of an optically active amine as aresolving agent or on a chiral HPLC column. Racemic mixtures can beseparated into their individual enantiomers by any of a number ofconventional methods. These include chiral chromatography,derivatization with a chiral auxiliary followed by separation bychromatography or crystallization, and fractional crystallization ofdiastereomeric salts.

Alternatively, any enantiomer of a compound of the general formula VIImay be obtained by stereospecific synthesis using optically purestarting materials or reagents of known configuration. In preferredembodiments, compounds of formula VII are administered asenantiomerically pure (or substantially enantiomerically pure)formulations.

Details for synthesizing pyrroles of formula VII of the invention areprovided in Example 12 below.

F. Combinations of a Statin Lactone and Another Active Agent

In another aspect, the present invention provides compositionscomprising combinations of a statin lactone with one or more additionalactive agents, preferably a pharmacologically active agent. Someembodiments include combinations comprising two or more statin lactones;two or more hydroxy acid forms of a statin; two or more non-statinanti-inflammatory agents; a statin lactone and a hydroxy acid form of astatin; a non-statin anti-inflammatory agent and a statin lactone; and anon-statin anti-inflammatory agent and a hydroxy acid form of a statin.In some embodiments, such combinations have molar ratios of about 99:1to about 1:99. Preferably, the range of molar ratios is selected fromabout 80:20 to about 20:80; about 75:25 to about 25:75, about 70:30 toabout 30:70, about 66:33 to about 33:66; about 60:40 to about 40:60;about 50:50; and about 90:10 to about 10:90. More preferably the molarratio is about 1:9, and most preferably about 1:1.

In some embodiments, a statin lactone is combined with the hydroxy acidform of the same or a different statin, or a pharmaceutically acceptablesalt thereof. In some embodiments, the statin lactone and hydroxy acid(or salt) forms may be combined in molar ratios of about 99:1 to about1:99. Preferably, the range of molar ratios of statin lactone: hydroxyacid (or salt) is selected from about 80:20 to about 20:80; about 75:25to about 25:75, about 70:30 to about 30:70, about 66:33 to about 33:66,about 60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90.More preferably the molar ratio of statin lactone: hydroxy acid (orsalt) is about 1:9, and most preferably about 1:1.

The statin lactone is preferably at least one lactone selected fromfluvastatin lactone, simvastatin lactone, lovastatin lactone,rosuvastatin lactone, pitavastatin lactone, glenvastatin lactone,cerivastatin lactone, pravastatin lactone, mevastatin lactone,bervastatin lactone and dalvastatin lactone, and most preferably isatorvastatin lactone. The statin hydroxy acid (or salt) is preferably atleast one acid (or salt) selected from fluvastatin, simvastatin,lovastatin, rosuvastatin, pitavastatin, glenvastatin, cerivastatin,pravastatin, mevastatin, bervastatin and dalvastatin, and mostpreferably is atorvastatin and/or pitavastatin. For example, in someembodiments, the calcium salt of atorvastatin and/or the sodium salt ofpitavastatin may be used. Still some embodiments use the sodium salt ofcerivastatin, also known as rivastatin; the sodium salt of fluvastatin;and/or nisvastatin, also referred to as NK-104. See, e.g., Drugs of theFuture, 1999, 24(5), pp. 511–513.

Additional details of compositions comprising a statin lactone combinedwith a hydroxy acid (or salt) form of a statin are provided in Example4. Specifically, Example 4a describes a gel formulation comprisingatorvastatin lactone and atorvastatin calcium, at a ratio of 1:1.Example 4b describes a tablet formulation comprising atorvastatinlactone and atorvastatin calcium, at a ratio of 1:1. Example 4cdescribes a tablet formulation comprising atorvastatin lactone andpitavastatin calcium, at a ratio of 1:1.

In some embodiments, a statin lactone is combined with a non-statinanti-inflammatory agent. For example, the statin lactone and non-statinanti-inflammatory agent may be combined in molar ratios of about 99:1 toabout 1:99. Preferably, the range of molar ratios of statin lactone:non-statin anti-inflammatory agent is selected from about 80:20 to about20:80; about 75:25 to about 25:75, about 70:30 to about 30:70, about66:33 to about 33:66; about 60:40 to about 40:60; about 50:50; and about90:10 to about 10:90. More preferably the molar ratio of statin lactone:non-statin anti-inflammatory agent is about 1:9, and most preferablyabout 1:1. The statin lactone is preferably at least one lactoneselected from fluvastatin lactone, simvastatin lactone, lovastatinlactone, rosuvastatin lactone, pitavastatin lactone, glenvastatinlactone, cerivastatin lactone, pravastatin lactone, mevastatin lactone,bervastatin lactone and dalvastatin lactone, and most preferably isatorvastatin lactone.

A non-statin anti-inflammatory agent can be any agent that is not astatin lactone or a hydroxy acid form of a statin or salt thereof.Preferably, the non-statin anti-inflammatory agent can be selected fromamong immunosuppressive agents, steroidal anti-inflammatory agents,anti-histamines, mast cell stabilizers and anti-autocoids, proteinkinase inhibitors, MAP kinase inhibitors, p38 MAP kinase inhibitors,disease-modifying anti-rheumatic drugs (DMARDs) and, more preferably,non-steroidal anti-inflammatory agents (NSAIDs).

Preferred immunosuppressive agents include, e.g., mycophenolate mofetil,cyclosporine, azathioprine, tacrolimus, pimecrolimus, sirolimus, and thelike. Preferred steroidal anti-inflammatory agents include, e.g.,prescription and nonprescription topical and aerosol corticosteroids,cortisone, cortisone acetate, hydrocortisone, dexamethasone,dexamethasone phosphate salts, prednisone, prednisolone phosphate salts,prednisolone, methyprednisolone, methylprednisolone acetate,beclomethasone, beclomethasone dipropionate, budesonide, ciclesonide,flunisolide, triamcinolone acetonide, and the like.

Preferred non-steroidal anti-inflammatory agents (NSAIDs) include, e.g.,salicylates, acetyl salicylic acid, sodium salicylate, colchicine,choline magnesium trisalicylate, salsalate, diflunisal, salicylsalicylicacid, sulfasalazine, olsalazine, indomethacin, sulindac, etodolac,macrolide immunosuppressives, tolmetin, clobetasol, dapsone,diflorasone, halobetasol, diclofenac, ketorolac, ibuprofen, naproxen,flurbiprofen, ketoprofen, fenoprofen, oxaprozin, mefanamic acid,meclofenamic acid, para-aminophenols, propionic acids, piroxicam,tenoxicam, meloxicam, nimesulide, phenylbutazone, oxyphenthatrazone,nabumetone, darbufelone, licofelone, rofecoxib, celecoxib, etoricoxib,valdecoxib, lumiracoxib, cimicoxib, parecoxib, BMS-347070, LAS-34475,GW-406,381, CS-501, P-54, FK-3311, S-2474, ajulemic acid, and the like,as well as pharmaceutically acceptable salts thereof. Other preferredNSAIDs include, e.g., prostaglandins, leukotrienes and thromboxanes, andother agents which inhibit cyclooxygenase enzymes, especially, specificcyclooxygenase inhibitors.

Additional details of compositions comprising a statin lactone combinedwith a non-statin anti-inflammatory agent are provided in Example 4.Specifically, Example 4d describes an ointment comprising atorvastatinlactone and the non-statin anti-inflammatory agent naproxen sodium, at aratio of 1:1. Example 4e describes an ointment comprising atorvastatinlactone and the non-statin anti-inflammatory agent diclofenac, at aratio of 1:1. Example 4f describes a rectal suppository comprisingatorvastatin lactone and the non-statin anti-inflammatory agentaminosalicylic acid, at a ratio of 1:9. Examples 4g, 4h, and 4i describean ointment, a gel and a cream, respectively, each comprisingatorvastatin lactone and the non-statin anti-inflammatory indomethacin,each at a ratio of 1:1.

In some embodiments of the present invention, a composition comprisingtwo or more of the aforementioned compounds, forms, and/or agentsprovides a synergistic effect. A synergistic effect can refer to one inwhich two or more compounds, forms and/or agents work together toproduce a total effect superior to and/or greater than that of any onecompound, form and/or agent alone. For example, the total effect may bea greater percentage increase in inhibition of a MAP kinase and/or agreater percentage increase in the inhibition of a MAP kinase-related invitro and/or in vivo effect and/or a greater percentage increase ineffectiveness in treating a MAP kinase-related condition. For example, asynergistic effect may be at least about a 5%, at least about a 10%, atleast about a 15%, at least about a 20%, at least about a 30%, at leastabout a 40%, at least about a 50%, at least about a 60%, at least abouta 70%, or at least about an 80% greater increase.

In preferred embodiments, combinations of a statin lactone and one ormore other active agents show a synergistic effect on reducing orinhibiting inflammation. For example, use of a combination of thepresent invention may provide a greater percentage decrease in swellingdue to inflammation compared to the effect using one or other ofcomponents of the combination alone. For example, in some embodiments, acombination of atorvastatin lactone with atorvastatin hydroxy acid (orsalt); atorvastatin lactone with pitavastatin hydroxy acid (or salt); oratorvastatin lactone with indomethacin produce synergistic effects onreducing or inhibiting inflammation, compared to the effect usingatorvastatin lactone alone, atorvastatin hydroxy acid (or salt) alone,pitavastatin hydroxy acid (or salt) alone, and/or indomethacin alone.Without being limited to a given hypotheis, theory and/or mechanism ofaction, the atorvastatin lactone may act by inhibiting p38α MAP kinase,centrally involved in inflammatory signaling cascades as discussedabove. Additional details or such combinations providing synergisticeffects on inflammation reduction or inhibition are provided in Example8 below, e.g., where treatment with formulations comprising acombination of atorvastatin lactone and the non-statin anti-inflammatoryagent indomethacin provides synergistic inhibitory effects oninflamamtion.

II. Methods of Treatment

Another aspect of the present invention relates to methods of usingpharmaceutical compositions and kits comprising compounds describedherein to treat kinase-related and/or reductase-related compounds,preferably MAP kinase-related and/or HMG-CoA reductase-relatedconditions, as well as novel uses of known compounds for the treatmentof MAP kinase-related conditions, and novel combinations for thetreatment of MAP kinase- and/or HMG CoA reductase-related conditions,especially inflammatory conditions.

The present invention provides methods, pharmaceutical compositions, andkits for the treatment of animal subjects. The term “animal subject” asused herein includes humans as well as other mammals. The term“treating” as used herein includes achieving a therapeutic benefitand/or a prophylactic benefit. By therapeutic benefit is meanteradication or amelioration of the underlying disorder being treated.For example, in an arthritic patient, therapeutic benefit includeseradication or amelioration of the underlying arthritis. Also, atherapeutic benefit is achieved with the eradication or amelioration ofone or more of the physiological symptoms associated with the underlyingdisorder such that an improvement is observed in the patient,notwithstanding the fact that the patient may still be afflicted withthe underlying disorder. For example, a MAP kinase inhibitor of thepresent invention provides therapeutic benefit not only when rheumatoidarthritis is eradicated, but also when an improvement is observed in thepatient with respect to other disorders or discomforts that accompanyrheumatoid arthritis, like stiffness or swelling in the joints.Similarly, compositions of the present invention can provide therapeuticbenefit in ameliorating other symptoms associated with MAPkinase-related conditions, e.g., inflammatory and/or autoimmuneconditions, including redness, rashes, swelling, itching, irritation,dryness, scaling, flaking, pain, temperature increase, loss of normalfunction, and the like.

For prophylactic benefit, a pharmaceutical composition of the inventionmay be administered to a patient at risk of developing a MAPkinase-related condition and/or a HMG-CoA reductase-related condition,or to a patient reporting one or more of the physiological symptoms ofsuch conditions, even though a diagnosis of the condition may not havebeen made. Administration may prevent the condition from developing, orit may reduce, lessen, shorten and/or otherwise ameliorate the conditionthat develops.

A. Treatment of MAP Kinase-related Conditions

The term “MAP kinase-related condition” as used herein refers to acondition in which directly or indirectly reducing the activity of aprotein kinase involved in signaling cascades of an allergic,inflammatory and/or an autoimmune response is desirable, and/or directlyor indirectly reducing the production and/or effects of one or moreproducts of the protein kinase is desirable. For example, a MAPkinase-related condition may involve over-production or unwantedproduction of one or more pro-inflammatory cytokines, such as tumornecrosis factor-α (TNF-α), interleukin-1β (IL-1β), or other chemicalmessengers of signal transduction pathways associated with inflammation(including responses to and expression of TNF-α and IL-1β), apoptosis,growth and differentiation.

Examples of MAP kinase-related conditions include but are not limited toallergic, inflammatory and autoimmune conditions, such as, for example,ocular allergic, ocular inflammatory and/or ocular autoimmuneconditions; allergic, inflammatory and/or autoimmune conditions of theear; allergic, inflammatory and/or autoimmune conditions of the skin andskin structures; gastrointestinal allergic, gastrointestinalinflammatory and/or gastrointestinal autoimmune conditions; respiratoryallergic, respiratory inflammatory and/or respiratory autoimmuneconditions; as well as arthritis, rheumatoid arthritis, and/or otherinflammatory/autoimmune diseases of the musculoskeletal system;osteoarthritis; vascular inflammatory conditions, vasculitis,inflammatory bowel disease (including ulcerative colitis and Crohn'sdisease), Celiac sprue, acne, psoriasis (as well as other papulosquamousdisorders such as lichen planus), topical dermatitis; atopic dermatitis(including eczema), irritant contact dermatitis, endotoxemia,restenosis, sepsis, and toxic shock syndrome, as well as transplantrejection. Other MAP kinase-related conditions include ageing,photo-ageing, cachexia, leprosy, Leishmaniasis, asthma, chronic pelvicpain, inflammatory muscle disease, allergic rhinitis (hay fever),gastritis, vaginitis, conjunctivitis, interstitial cystitis, chronicfatigue syndrome, osteoporosis, scleroderma, and the like. MAP-kinaserelated conditions can also include diabetes, chronic obstructivepulmonary disease, as well as cardiovascular-related conditions such asatherosclerosis, myocardial infarction, congestive heart failure,ischemic-reperfusion injury and other vascular inflammatory conditions.MAP-kinase related conditions can also include proliferative disorders,including cancers, e.g., multiple myeloma, fibrotic disorders, mesangialcell proliferative disorders, such as glomerulonephritis, diabeticnephropathy malignant nephrosclerosis, thrombotic microangiopathysyndromes, organ transplant rejection and glomerulopathies. MAP-kinaserelated condition can also include neurodegenerative diseases, e.g.Alzheimer's and pain sensation, as well as infectious diseases such asviral, bacterial, and fungal infections.

Other conditions treatable with compositions, kits, and methods of thepresent invention include those currently treated with soluble TNFreceptors, anti-TNF antibodies, IL-1 receptor antagonists, TNF-αconverting enzyme inhibitors, inhibitors of protein-tyrosine kinasesand/or inhibitors of protein serine/threonine kinases of the MAP kinasefamily, preferably including conditions currently treated withinhibitors of p38 MAP kinases and/or the stress-activated proteinkinases/Jun N-terminal kinases (SAPKs/JNKs). Most preferably, conditionstreatable with the practice of this invention include those relating top38α MAP kinase, e.g, conditions currently treated by inhibition of p38αMAP kinase activity.

Reducing the activity of a protein kinase, e.g. a MAP kinase, is alsoreferred to as “inhibiting” the kinase. The term “inhibits” and itsgrammatical conjugations, such as “inhibitory,” do not require completeinhibition, but refer to a reduction in kinase activity. Such reductionis preferably by at least about 50%, at least about 75%, at least about90%, and more preferably by at least about 95% of the activity of theenzyme in the absence of the inhibitory effect, e.g., in the absence ofan inhibitor. Conversely, the phrase “does not inhibit” and itsgrammatical conjugations refer to situations where there is less thanabout 20%, less than about 10%, and preferably less than about 5%, ofreduction in enzyme activity in the presence of the compound. Furtherthe phrase “does not substantially inhibit” and its grammaticalconjugations refer to situations where there is less than about 30%,less than about 20%, and preferably less than about 10% of reduction inenzyme activity in the presence of the compound.

The ability to reduce enzyme activity is a measure of the potency or theactivity of a compound, or combination of compounds, towards or againstthe enzyme. Potency is preferably measured by cell free, whole celland/or in vivo assays in terms of IC50, K_(i) and/or ED50 values. AnIC50 value represents the concentration of a compound required toinhibit enzyme activity by half (50%) under a given set of conditions. AK_(i) value represents the equilibrium affinity constant for the bindingof an inhibiting compound to the enzyme. An ED50 value represents thedose of a compound required to effect a half-maximal response in abiological assay. Further details of these measures will be appreciatedby those of ordinary skill in the art, and can be found in standardtexts on biochemistry, enzymology, and the like.

In some embodiments, compounds in one or more forms represented byformulas I, II, III, and IV inhibit a MAP kinase. These compounds canexert anti-inflammatory effects in vitro and/or in vivo and can form thebasis for pharmaceutical compositions useful in the treatment of MAPkinase-related conditions, e.g., allergic, inflammatory and/orautoimmune diseases, in humans and other mammals. In certainembodiments, for example, these compositions reduce production of, andsignaling pathways involving, TNF-α and IL-1β.

As noted above, a subset of the compounds of formulas I and II are novelanalogs of known inhibitors of MAP kinases, wherein X comprises alipophilic MAP kinase inhibitor or a lipophilic moiety or analogthereof. Some of these analogs retain MAP kinase inhibitory activity inthe lactone and/or acid forms, and are useful in the practice of thisinvention, e.g. in a method of treating a MAP kinase-related conditionby administering to a subject an effective amount of at least one ofsuch compounds. For example, certain lactone derivatives illustrated inFIG. 5 are preferred in some embodiments, as described in detail above.

Also as noted above, a subset of the compounds of formulas I and II arenovel analogs of known inhibitors of HMG-CoA reductase, wherein Xcomprises a lipophilic moiety of an HMG-CoA reductase inhibitor, e.g., astatin, a synthetic statin, or an analog thereof. Some of these analogsdisplay MAP kinase inhibitory activity in the lactone and/or acid forms,and are useful in the practice of this invention, e.g. in a method oftreating a MAP kinase-related condition by administering to a subject aneffective amount of at least one of such compounds. For example, certainlactone derivatives illustrated in FIG. 7 are preferred in someembodiments, as described in detail above, while the specific lactonederivatives illustrated in FIG. 8 are even more preferred. In someembodiments, the acid forms of such compounds also display MAP kinaseinhibitory activity. In some preferred embodiments, MAP kinaseinhibition is not reversed by addition of farnesyl pyrophosphate,geranyl geranyl pyrophosphate, mevalonate or any downstream product ofmevalonate. In some embodiments, the lactone form does not inhibit ordoes not substantially inhibit HMG-CoA reductase. In some preferredembodiments, a lactone form may be formulated into solutions,suspensions, ointments and/or suppositories for topical applicationand/or rectal administration. The formulation may be applied to regionsof inflammation, e.g., to exert a local effect, as described in moredetail below. Preferably, in such embodiments, little or no systemiceffects are observed

Another subset of the compounds of formula I are known inhibitors ofHMG-CoA reductase, including mevasatin, lovastatin, simvastatin,pravastatin, fluvastatin, atorvastatin, cerivastatin, rosuvastatin,pitavastatin, glenvastatin, bervastatin, dalvastatin, eptastatin,dihydroeptastatin, itavastatin, L-154819, advicor, L-654969, and otherstatin drugs used to treat disorders such as hypercholesterolemia.

In the case of these compounds, the present invention relates to the useof the corresponding lactones of formula I in treating MAPkinase-related conditions, e.g., inflammatory diseases, in particularconditions where inhibiting p38α MAP kinase activity is desirable andwhere a synthetic statin lactone is used. For instance, such compoundsfind use in treating an inflammatory condition by administering aneffective amount of a statin lactone to a subject where the lactoneinhibits a MAP kinase. For example, the statin lactones illustrated inFIG. 6 are preferred in some embodiments for treating MAP kinase-relatedconditions. More preferred statin lactones are those derived fromatorvastatin, fluvastatin, rosuvastatin, cerivastatin, pitavastatin andglenvastatin. In some preferred embodiments, MAP kinase inhibition isnot reversed by addition of farnesyl pyrophosphate, geranyl geranylpyrophosphate, mevalonate or any downstream product of mevalonate. Insome preferred embodiments, the lactone is not hydrolyzed, or notsubstantially hydrolyzed, to an acid form. In some such embodiments, thelactone does not inhibit or does not substantially inhibit HMG-CoAreductase. In some of these preferred embodiments, a lactone form may beformulated into solutions, suspensions, ointments and/or suppositoriesfor topical application and/or rectal administration. The formulationmay be applied to regions of inflammation, e.g., to exert a localeffect, as described in more detail below. Preferably, in suchembodiments, little or no systemic effects are observed. In someembodiments, the acid forms of such compounds also display MAP kinaseinhibitory activity.

TABLE I Compound IC50 p38α Atorvastatin calcium >100 μM Atorvastatinlactone 20 μM Fluvastatin sodium 34 μM Fluvastatin lactone 45 μMRosuvastatin calcium 92 μM Rosuvastatin lactone >100 μM

The δ-lactone forms of atorvastatin and fluvastatin show inhibitoryactivity against human p38α MAP kinase. Atrovastatin lactone gave anIC50 of about 20 μM, while fluvastatin lactone exhibited an IC50 valueof about 45 μM in some embodiments. Also, the acid/salt form offluvastain (e.g., fluvastatin sodium) inhibits p38α MAP kinase, showingan IC50 of about 34 μM in some embodiments.

Further, certain lipophilic MAP kinase inhibitors as well as lipophilicmoieties and analogs thereof, having structures that favor a closed ringstructure or cyclic form, including compounds of formulas III and IVdescribed above, can also display MAP kinase inhibitory activity.Structures comprising a lipophilic MAP kinase inhibitor or a lipophilicmoiety thereof, for example the MAP kinase inhibitors of FIG. 4, as wellas lipophilic moieties or analogs thereof, such as those of FIG. 5, arepreferred in some embodiments.

Further, certain analogs of known lipophilic HMG-CoA reductaseinhibitors having structures modified to favor a closed ring structureor cyclic form, including compounds of formulas III and IV describedabove, can also display MAP kinase inhibitory activity. Such structurescan be useful in the practice of this invention, e.g., in a method oftreating a MAP kinase-related condition by administering to a subject aneffective amount of at least one of such compounds. In some embodiments,a compound of formula III or IV does not inhibit or does notsubstantially inhibit HMG-CoA reductase. Structures comprising a statinor a lipophilic moiety of a statin, for example, the statins of FIG. 6,as well as lipophilic moieties of statin analogs, such as those of FIGS.7 and 8, are preferred in some embodiments. More preferred embodimentsinclude des-oxo and δ-lactam derivatives from a synthetic statin, ordes-oxo and δ-lactam derivatives from atorvastatin, fluvastatinrosuvastatin, cerivastatin, pitavastatin and glenvastatin, as describedabove.

The present invention also includes kits that can be used to treat a MAPkinase-related conditions. These kits comprise a compound or combinationof compounds described herein and preferably instructions teaching theuse of the kit according to the various methods and approaches describedherein. Such kits also include information, such as scientificliterature references, package insert materials, clinical trial results,and/or summaries of these and the like, which indicate or establish theactivities and/or advantages of the compound. Such information may bebased on the results of various studies, for example, studies usingexperimental animals involving in vivo models and studies based on humanclinical trials. Kits described herein can be provided, marketed and/orpromoted to health providers, including physicians, nurses, pharmacists,formulary officials, and the like.

B. Treatment of HMG-CoA Reductase-related Conditions

The term “HMG-CoA reductase-related condition” as used herein refers toa condition in which directly or indirectly reducing the activity ofHMG-CoA reductase is desirable and/or directly or indirectly reducingthe production and/or effects of one or more products of HMG-CoAreductase is desirable. For example, an HMG-CoA reductase-relatedcondition may involve elevated levels of cholesterol, in particular,non-HDL cholesterol in plasma, such as elevated levels of LDLcholesterol. Typically, a patient is considered to have high or elevatedcholesterol levels based on a number of criteria, for example, seePearlman, Postgrad. Med. 112(2):13–26 (2002), incorporated herein byreference. Guidelines include serum lipid profiles, such as LDL comparedwith HDL levels.

Examples of HMG-CoA reductase-related conditions includehypercholesterolemia, lipid disorders such as hyperlipidemia, andatherogenesis and its sequelae of cardiovascular diseases, includingatherosclerosis, other vascular inflammatory conditions, myocardialinfarction, ischemic stroke, occlusive stroke, and peripheral vasculardiseases, as well as other conditions in which decreasing cholesteroland/or other products of the cholesterol biosynthetic pathways canproduce a benefit. Other HMG-CoA reductase-related conditions treatablewith compositions, kits, and methods of the present invention includethose currently treated with statins.

Reducing the activity of HMG-CoA reductase, is also referred to as“inhibiting” the enzyme. The term “inhibits” and its grammaticalconjugations, such as “inhibitory,” do not require complete inhibition,but refer to a reduction in HMG-CoA reductase activity. Such reductionis preferably by at least about 50%, at least about 75%, at least about90%, and more preferably by at least about 95% of the activity of theenzyme in the absence of the inhibitory effect, e.g., in the absence ofan inhibitor. Conversely, the phrase “does not inhibit” and itsgrammatical conjugations refer to situations where there is less thanabout 20%, less than about 10%, and preferably less than about 5% ofreduction in enzyme activity in the presence of the compound. Furtherthe phrase “does not substantially inhibit” and its grammaticalconjugations refer to situations where there is less than about 30%,less than about 20%, and preferably less than about 10% of reduction inenzyme activity in the presence of the compound.

The ability to reduce enzyme activity is a measure of the potency or theactivity of the compound or combination of componds towards or againstthe enzyme. Potency is preferably measured by cell free, whole celland/or in vivo assays in terms of IC50 or ED50 values. An IC50 valuerepresents the concentration of a compound required to inhibit theenzyme activity by half (50%) under a given set of conditions. A Kivalue represents the equilibrium affinity constant for the binding of aninhibiting compound to the enzyme. An ED50 value represents the dose ofa compound required to effect a half-maximal response in a biologicalassay. Further details of these measures will be appreciated by those ofordinary skill in the art, and can be found in standard texts onbiochemistry, enzymology, and the like.

In some embodiments, compounds in one or more forms represented byformulas I, II, III, and IV inhibit HMG-CoA reductase. In manyembodiments, compounds of formula II inhibit HMG-CoA reductase. Suchcompounds find use in the practice of this invention e.g., in a methodof treating an HMG-CoA reductase-related condition by administering to asubject an effective amount of at least one of such compounds. Thesecompounds can lower cholesterol levels in vitro and in vivo, and/orincrease HDL, thereby forming the basis for pharmaceutical compositionsuseful in the treatment of HMG-CoA reductase-related conditions, e.g.,hypercholesterolemia and atherosclerosis, in humans and other mammals.

As noted above, a subset of the compounds of formulas I and II are novelanalogs of known inhibitors of MAP kinases, wherein X comprises alipophilic MAP kinase inhibitor or a lipophilic moiety or analogthereof. Some of these analogs display HMG-CoA reductase inhibitoryactivity, for example, in the acid form (formula II), and are useful inthe practice of this invention, e.g., in a method of treating an HMG-CoAreductase-related condition by administering to a subject an effectiveamount of at least one of such compounds. For example, acid forms, inparticular the carboxylate forms, of certain lactone derivativesillustrated in FIG. 5 are preferred in some embodiments for thetreatment of HMG-CoA reductase-related conditions.

Also as noted above, a subset of the compounds of formulas I and II arenovel analogs of known inhibitors of HMG-CoA reductase, wherein Xcomprises a lipophilic moiety of an HMG-CoA reductase inhibitor, e.g., astatin, or an analog thereof. Some of these analogs retain HMG-CoAreductase inhibitory activity in the lactone and/or acid form, inparticular, in the acid carboxylate form, and are useful in the practiceof this invention, e.g., in a method of treating an HMG-CoAreductase-related condition by administering to a subject an effectiveamount of at least one of such compounds. For example, acid carboxylateforms of certain lactone derivatives illustrated in FIG. 7 are preferredin some embodiments.

Also as noted above, in some embodiments, a compound of the instantinvention, or a composition comprising one or more such compounds, canbe used in treating an HMG-CoA reductase-related condition by increasingHDL levels. Higher levels of HDL are believed to protect against, e.g.,atherosclerosis, whereas low HDL is recognized as an independent riskfactor for cornary artery disease. For example, an HDL level below about40 mg/DL can be considered in need of treatment. Without being limitedto a particular theory and/or hypothesis, compounds of the instantinvention can bring about an upregulation of HDL in treating an HMG Co-Areductase related condition, such as artherosclerosis.

Upregulating HDL is also referred to as “increasing” HDL or HDL levels.The term “increases” and its grammatical conjugations can refer to asmall, significant and/or substantial increase, preferably an increasesufficient to decrease a risk of an HMG-CoA reductase-related conditionin a subject being treated. Such increase is preferably by at leastabout 10%, at least about 20%, at least about 30%, and more preferablyby at least about 50% of HDL levels in the absence of treatment. Inpreferred embodiments, atorvastatin and analogs of atorvastatin are usedto increase HDL.

The present invention also includes kits that can be used to treat anHMG-CoA reductase-related condition. These kits comprise a compound orcombination of compounds described herein, and preferably instructionsteaching the use of the kit according to the various methods andapproaches described herein. Such kits also include information, such asscientific literature references, package insert materials, clinicaltrial results, and/or summaries of these and the like, which indicate orestablish the activities and/or advantages of the compound(s). Suchinformation may be based on the results of various studies, for example,studies using experimental animals involving in vivo models and studiesbased on human clinical trials. Kits described herein can be provided,marketed and/or promoted to health providers, including physicians,nurses, pharmacists, formulary officials, and the like.

C. Treatment of both MAP kinase- and HMG-CoA Reductase-relatedConditions

One of the purposes of this invention is to describe compounds orcombinations of compounds which inhibit both MAP kinase and HMG-CoAreductase. Such compounds or combinations can exert concomitantanti-inflammatory and cholesterol-lowering effects in vitro and/or invivo. In certain embodiments, for example, these compounds orcombinations reduce production of, and signaling pathways involving,TNF-α and IL-1β, as well as inhibiting production of cholesterol and/orother downstream products of mevalonate, including mevalonatepyrophosphate, isopentyl pyrophosphate, geranyl pyrophosphate, farnesylpyrophosphate, dolichols, farnesylated proteins, trans-transgeranylgeranyl pyrophosphate, ubiquinone, geranyl-geranylated proteins,squalene, and the like. Further, in some embodiments, these compounds orcombinations can exert superior anti-atherogenesis and/oranti-inflammatory effects in vivo.

Such compounds or combinations can form the basis for pharmaceuticalcompositions, kits, and methods for treating both MAP kinase-relatedconditions and HMG-CoA reductase-related conditions in humans and otheranimals. Moreover, such compositions can provide superior benefits intreating HMG-CoA reductase-related conditions, such as cardiovasculardisease, compared with treatments that inhibit HMG-CoA reductase but donot inhibit or do not substantially inhibit MAP kinase. Also,compositions of the present invention can provide superior benefits intreating MAP kinase-related conditions, such as inflammatory conditions,compared with treatments that inhibit MAP kinases but do not inhibit ordo not substantially inhibit HMG-CoA reductase.

FIG. 10, for example, illustrates a treatment approach in whichcompositions of the present invention produce a benefit in both MAPkinase- and HMG-CoA reductase-related conditions. This figure servesonly as an example, and is in no way intended to be limiting withrespect to the present invention. For example, those skilled in the artwill readily appreciate variations and modifications of the schemeillustrated, and such variations and modifications are also contemplatedas being contained within the scope of the invention.

As FIG. 10 illustrates, the δ-lactone form (formula I) of a compound ofthis invention can inhibit a MAP kinase, and the acid form (formula II),in particular the deprotonated carboxylate form (formula IIb), caninhibit HMG-CoA reductase. Accordingly, this treatment approach canprovide a benefit in both a HMG-CoA reductase-related condition and aMAP kinase-related condition, for instance, in a method comprisingadministering to a subject an effective amount of at least one of suchcompounds, e.g., reducing pro-inflammatory cytokine production, in thetreatment of a MAP kinase-related condition, such as an allergic,inflammatory and/or autoimmune condition, and reducing cholesterolproduction in the treatment of a HMG-CoA reductase-related condition,such as cardiovascular disease. This reduction in pro-inflammatorycytokine production by inhibition of a MAP kinase, e.g., p38α MAPkinase, may be in addition to other immunomodulatory effects of someHMG-CoA reductase inhibitors that may, for example, produceimmunomodulatory responses though the action of metabolites such asfarnesyl pyrophosphate and/or geranylgeranyl pyrophosphate. Moreover, insome embodiments, the role of a compound of the present invention in aMAP kinase-related pathway is distinct from the anti-inflammatoryeffects of some statins through metabolite products such asgeranylgeranyl pyrophosphate and/or farnesyl pyrophosphate. For example,the inhibitory activity of some compounds of this invention on a MAPkinase and on MAP-kinase related conditions need not be reversed byexogenous addition of mevalonate (e.g., sodium mevalonate),geranylgeranyl pyrophosphate, farnesyl pyrophosphate, and/or otherdownstream product of mevalonate.

Furthermore, the interplay between inflammatory and HMG-CoAreductase-related disorders means that compositions regulating both aMAP kinase and HMG-CoA reductase pathways can be particularlybeneficial. Inhibition of HMG-CoA reductase can lead to improved serumlipid profiles, such as decreased LDL and increased HDL levels, which inturn can lead to a reduction in the rate of atherogenesis. On the otherhand, initiation of atherogenic plaque deposition (e.g., via foam cells)is reduced by the anti-inflammatory effects, including those whichderive from inhibition of a MAP kinase. Inhibition of a MAP kinase canalso antagonize inflammatory processes which contribute to thedisruption of atherogenic plaques and which, in turn, can lead toarterial thrombosis, blockade, etc. Consequently, pharmaceuticalcompositions including a compound of formula I/II and having inhibitoryactivity against both a MAP kinase and HMG-CoA reductase can besuperior, preferably differentially superior, to drugs targeting onlyHMG-CoA reductase. In some preferred embodiments, such compositions canprovide a differentially superior benefit in treating cardiovasculardisease related to atherogenesis, including formation and disruption ofatherosclerotic plaques. Further, pharmaceutical compositions includinga compound or combination of compounds of formula I and/or formula IIhaving inhibitory activity against both a MAP kinase and HMG-CoAreductase can be superior, preferably differentially superior, to drugstargeting only a MAP kinase in terms of treating a MAP kinase-relatedcondition, such as inflammation, again due to the interplay betweeninflammatory and cardiovascular conditions.

In some embodiments, a compound of formula I inhibits or is more potentagainst a MAP kinase while the corresponding compound of formula II,with equivalent X, Y, Z, A and stereochemistry, inhibits or is morepotent against HMG-CoA reductase. In some preferred embodiments, theactivities or potencies of a compound of formula I and the correspondingcompound of formula II are similar towards a MAP kinase and HMG-CoAreductase. In preferred embodiments, the potencies of these formsagainst their respective targets differs by no more than a factor ofabout 1000, more preferably about 100, and most preferably about 10. Ina preferred embodiment, compounds of formulas I and II have absoluteconfiguration as illustrated in FIG. 3 b and designated as (T,T)absolute configuration, as defined above.

In other preferred embodiments, the potency of a compound of formula Iand/or II against a MAP kinase is greater than its potency againstHMG-CoA reductase. In such embodiments, potencies with respect to a MAPkinase and HMG-CoA reductase differ by at least a factor of about 10.More preferably, potencies can differ by more than a factor of about100. Most preferably, potencies can differ by more than a factor ofabout 1000. In yet other preferred embodiments, the potency of acompound of formula I and/or II against HMG-CoA reductase is greaterthan its potency against a MAP kinase. In such embodiments, potencieswith respect to HMG-CoA reductase and a MAP kinase differ by at least afactor of about 10. More preferably, potencies can differ by more than afactor of about 100. Most preferably, potencies can differ by more thana factor of about 1000.

In some embodiments, a compound of formula I inhibits both a MAP kinaseand HMG-CoA reductase. In some embodiments, a compound of formula IIinhibits both a MAP kinase and HMG-CoA reductase. In other embodiments,a compound of formula III inhibits both a MAP kinase and HMG-CoAreductase; in still other embodiments, a compound of formula IV inhibitsboth a MAP kinase and HMG-CoA reductase. In nearly all preferredembodiments, compounds of formula II inhibit HMG-CoA reductase.

As noted above, a subset of the compounds of formulas I and II are novelanalogs of known inhibitors of MAP kinases, wherein X comprises alipophilic MAP kinase inhibitor or a lipophilic moiety or analogthereof. Some of these analogs retain MAP kinase inhibitory activity inthe lactone and/or acid forms while also exhibiting HMG-CoA reductaseinhibitory activity in the acid and/or lactone forms. In some preferredembodiments, a MAP kinase analog of the present invention inhibits a MAPkinase in the lactone form of formula I and inhibits HMG-CoA reductasein the corresponding acid form of formula II, in particular thecarboxylate form of formula IIb. In preferred embodiments, suchcompounds are useful in the present invention, e.g., in a methodcomprising administering to a subject an effective amount of at leastone of such compounds to treat a MAP kinase-related condition andadditionally treating an HMG-CoA reductase-related condition. MAP kinaseanalogs illustrated in FIG. 5 can provide examples of such preferredembodiments. In other embodiments, the lactone form inhibits both a MAPkinase and HMG-CoA reductase; in still other embodiments, the acid forminhibits both a MAP kinase and HMG-CoA reductase.

Also as noted above, a subset of the compounds of formulas I and II arenovel analogs of known inhibitors of HMG-CoA reductase, wherein Xcomprises a lipophilic moiety of an HMG-CoA reductase inhibitor, e.g., astatin, a synthetic statin, or an analog thereof. Some of these analogsretain HMG-CoA reductase inhibitory activity in the acid and/or lactoneforms while also exhibiting MAP kinase inhibitory activity in thelactone and/or acid forms. In some preferred embodiments, a statinanalog of the present invention inhibits HMG-CoA reductase in the acidform of formula II (in particular, in the carboxylate form of formulaIIb) and inhibits a MAP kinase in the corresponding lactone form offormula I. In preferred embodiments, such compounds find use in thepractice of the invention, e.g., in a method comprising administering toa subject an effective amount of at least one of such compounds to treata MAP kinase-related condition and additionally treating an HMG-CoAreductase-related condition. Statin analogs illustrated in FIG. 7 canprovide examples of such preferred embodiments, and the specificexamples illustrated in FIG. 8 can provide even more preferredembodiments. In other embodiments, the lactone form inhibits both a MAPkinase and HMG-CoA reductase; in still other embodiments, the acid forminhibits both a MAP kinase and HMG-CoA reductase.

The present invention also includes kits that can be used to treat MAPkinase- and HMG-CoA reductase-related conditions, in particularcardiovascular disease related to atherogenesis. These kits can comprisea compound or combination of compounds described herein, includingcompounds of formula I and/or II which have inhibitory activity againstboth a MAP kinase and HMG-CoA reductase, and preferably instructionsteaching the use of the kit according to the various methods andapproaches described herein.

Such kits also include information, such as scientific literaturereferences, package insert materials, clinical trial results, and/orsummaries of these, and the like, which indicate or establish themultiple activities of the compounds or combination and indicate and/orestablish how its use provides advantages and/or differentialsuperiority in treating an HMG-CoA reductase- and/or a MAPkinase-related condition, preferably in treating cardiovascular disease.Such information may be based on the results of various studies, forexample, studies using experimental animals involving in vivo models andstudies based on human clinical trials. Kits of the present inventionmay also include materials comparing the approaches of the presentinvention with other therapies, which do not display a combination ofMAP kinase plus HMG-CoA reductase inhibitory activities. Kits describedherein can be provided, marketed and/or promoted to health providers,including physicians, nurses, pharmacists, formulary officials, and thelike.

D. Treatment of Inflammatory Conditions

Another aspect of the present invention relates to methods of usingpharmaceutical compositions and kits comprising combinations ofcompounds, forms and/or agents described herein to treat MAPkinase-related and/or HMG-CoA reductase-related conditions that areinflammatory conditons. Inflammatory conditions, as used herein, canrefer to inflammatory diseases or disorders associated with inflammationdue to relatedness to MAP kinase-, HMG CoA reductase- and/or otherpathways. Inflammatory conditions treatable using some embodiments ofthe instant invention can involve different organ systems, and can varyin severity from trivial to lethal.

For example, inflammatory conditions of the skin can be treated in someembodiments of the instant invention, including, but not limited to,atopic dermatitis, age-related effects, acne, eczema, psoriasis and skincancer. Various types of acne can be treated uisng some embodiments ofthe instant invention, including, e.g., acne atrophica, bromide orchlorine acne, common acne (acne vulgaris), acne conglobata, contactacne, contagious acne of horses, acne cosmetica, cystic acne, acnedetergicans, epidemic acne, acne estivalis, excoriated acne, acnefrontalis, acne fulminans, halogen acne, acne indurata, infantile acne,iodide acne, acne keloid, acne mechanica, acne necrotica miliaris,neonatal acne, acne papulosa, picker's acne, pommade acne, premenstrualacne, acne pustulosa, acne rosacea, acne scorbutica, acne scrofulosorum,acne tropicalis, acne urticata, acne varioliformis, acne venenata, andthe like.

Various types of eczema can be treated in some embodiments of theinstant invention, including, e.g., eczematous dermatitis, such asatopic dermatitis, the most common form of eczema, generally seen ininfants and young adults. Eczema can present as a red, itchy,non-contagious inflammation of the skin that can be acute or chronic,possibly accompanied by red skin patches, pimples, crusts, scabs, andwatery discharge.

Various effects of ageing can be treated in some embodiments of theinstant invention, including, e.g., skin-related inflammatory diseasesattributable to ageing. Such effects can include formation of wrinklesand fine lines, slackening of cutaneous and subcutaneous tissue, loss ofskin elasticity, reduction in skin tone and texture and/or yellowing.Loss of elasticity can result form atrophy of the epidermis, beginningon a small scale and eventually decreasing the number of cells in thedermis. Capillaries can become more susceptible to bruising, collagenmetabolism may slow, and/or the concentration of the cell surfacemolecule glycosaminoglycan (believed to have a role in the recognitionof other cells and substrates) may decrease. With ageing, skin mayexhibit chronic inflammation with enlarged fibroblasts. Effects ofageing aggravated with sun exposuse can also be treated in someembodiments, e.g., pigmentation marks, telangiectasias, elastosis,and/or other skin photo-damage, as well as benign, premalignant and/ormalignant neoplasms (e.g., caused by prolonged sun exposure). Ageingitself, e.g., can be considered as an inflammatory condition, e.g., asthe ability to mount an inflammatory response decreases and healing timefor injuries increases with age.

Inflammatory conditions of the skin that can be treated in someembodiments of the instant invention include skin cancers and otherhyperproliferative skin disorders, including, e.g., without being notlimited to, basal cell carcinoma, squamous cell carcinoma (Bowen'sdisease), keratosis (such as actinic or seborrheic keratosis), and/ordisorders of keratinization (such as ichthyosis and keratoderma).

Inflammatory conditions of the respiratory system can be treated in someembodiments of the instant invention, including, but not limited to,allergic rhinitis, chronic obstructive pulmonary disease, adultrespiratory distresss syndrome, asthma, and the like. Allergic asthmacan include atopic, chronic diseases of the lung characterized byinflammation of the air passages. Allergic rhinitis or hay fever caninclude conditions that affect mucous membranes characterized byseasonal or perennial nasal inflammation, e.g., in response to anallergen. Other mucous inflammatory conditions treatable using someembodiments of the instant invention can include lamellar ichthyosis,acne, rosacea, and the like.

Inflammatory conditions of the urogenital tract can be treated in someembodiments of the instant invention, including, but not limited to,vaginitis and interstitial cystitis, and other conditions characterizedby inflammation of the urogenital epithelium and/or the urinary bladder.Interstitial cystitis also can include other conditions associated witha dysfunctional bladder glycosaminoglycan protective layer and/orincreased numbers of activated bladder mast cells.

Inflammatory conditions of the gastrointestinal tract can be treated insome embodiments of the instant invention, including, but not limitedto, celiac diseases, e.g., celiac sprue, inflammatory bowel disease(including ulcerative colitis and Crohn's disease), intestinalinfections, enterocolitis, gastritis, and the like.

Inflammatory conditons of the musculoskeletal system can be treated insome embodiments of the instant invention, including, but not limitedto, inflammatory muscle pain, arthritis, rheumatoid arthritis, psoriaticarthritis, osteoarthritis, osteoporosis, and the like. Osteoporosis caninclude conditions characterized by decreased bone mass, increasedfragility of the remaining bone, and/or increased incidence offractures.

Inflammatory conditions of the vascular system an be treated in someembodiments of the instant invention, including, but not limited tohypercholesterolemia, hyperlipidemia, atherogenesis and associatedcardiovascular risks of atherosclerosis, thrombosis, myocardialinfarction, ischemic stroke, ischemic-reperfusion injury, peripheralvascular disease, e.g., peripheral occlusive disease, and the like.Inflammatory conditions of the systemic circulation also includeendotoxemia, lupus erythrematosus, sepsis, toxic shock syndrome andtransplant rejection, and may also be treated in some embodiments.

Inflammatory conditions of the central nervous system can be treated insome embodiments of the instant invention, including, but not limitedto, neurogenic inflammation and neurodegenerative diseases, such asAlzheimer's disease, and the like.

In some embodiments, compositions comprisng combinations of compunds,forms and/or agents described herein provide treatments for autoimmuneconditions. Autoimmune conditions as used herein can include organ ortissue-specific autoimmune conditions, as well as those which affect thewhole body. Organ or tissue-specific autoimmune conditions that can betreated in some embodiments of the instant invention include, e.g., typeI diabetes mellitus, multiple sclerosis, primary billiary cirrhosis,Hashimotos thyroiditis, pernicious anemia, Crohn's disease, Addison'sdisease, myasthenia gravis, rheumatoid arthritis, uveitis, psoriasis,Guillain-Barre Syndrome, Graves' disease, and the like. Systemicautoimmune conditions that can be treated in some embodiments include,e.g., systemic lupus erythematosus, ermatomyositis, and the like.

As detailed above, in some embodiments, combinations comprising a statinlactone and one or more additional active agents can be used in treatingone or more of the inflammatory conditions provided herein. Preferredcombinations can depend on the affected system. For example,combinations comprising a statin lactone and a salt form of a hydroxyacid statin are preferred in the treatment of inflammatory conditions ofthe vascular system and central nervous system, especially Alzheimer'sdisease, as well as inflammatory conditions of the skin, especiallyeczema, psoriasis, acne and the effects of ageing. Also as detailedabove, preferred combinations comprise atorvastatin lactone with a saltof either atorvastatin or pitavastatin in a molar ratio of about 90:10to about 10:90. In other embodiments, combinations comprising a statinlactone and a non-statin anti-inflammatory agent are preferred, e.g.,where combinations comprising atorvastatin lactone and a non-steroidalanti-inflammatory drug are used in a molar ratio of about 90:10 to about10:90.

One of the purposes of this invention is to teach combinations ofcompounds that can produde synergistic effects in treating aninflammatory condition, e.g., one or more of the inflammatory conditionsprovided herein. For example, in some prefered embodiments, acombination of a statin lactone and another active agent provides asynergistic effect in treating an inflammatory condition, as detailedabove. Additional details of combinations providing synergisticinhibitory effects in treating inflammation are provided in Example 8below.

III. Formulations, Routes of Administration, and Effective Doses

Yet another aspect of the present invention relates to formulations,routes of administration and effective doses for pharmaceuticalcompositions comprising a compound or combination of compounds of theinstant invention. Such pharmaceutical compositions can be used to treatinflammatory, MAP kinase-related, and/or HMG-CoA reductase-relatedconditions, as described in detail above.

The compounds of formula I/II may be provided in a either the lactone oracid form, and/or may be allowed to interconvert in vivo afteradministration. That is, either the δ-lactone or hydroxy carboxylic acidform, or pharmaceutically acceptable salts, esters or amides thereof,may be used in developing a formulation for use in the presentinvention. Further, in some embodiments, the compound may be used incombination with one or more other compounds or with one or more otherforms. For example a formulation may comprise both the lactone and acidforms in particular proportions, depending on the relative potencies ofthe lactone and acid forms and the intended indication. For example, incompositions for treating both MAP kinase- and HMG-CoA reductase-relatedconditions where the lactone form inhibits MAP kinase and the acid(carboxylate) form inhibits HMG-CoA reductase, and where potencies aresimilar, about a 1:1 ratio of lactone to acid forms may be used. The twoforms may be formulated together, in the same dosage unit e.g. in onecream, suppository, tablet, capsule, or packet of powder to be dissolvedin a beverage; or each form may be formulated in a separate unit, e.g.,two creams, two suppositories, two tablets, two capsules, a tablet and aliquid for dissolving the tablet, a packet of powder and a liquid fordissolving the powder, etc.

Similarly, compounds of formula III and IV, or their pharmaceuticallyacceptable salts, esters, or amides thereof, may be used alone,together, or in combination with the corresponding or other compounds offormula I and II, described above. For example, a compound of formula IV(closed δ-lactam ring) may be co-administered with a compound of formulaII (open acid form), where the compounds have equivalent X, Y, Z, A andstereochemistries. Such administration may be useful for treating bothMAP kinase- and HMG-CoA reductase-related conditions, for example, wherethe lactam form inhibits MAP kinase and the acid (carboxylate) forminhibits HMG-CoA reductase. The two forms may be formulated together, inthe same dosage unit e.g. in one cream, suppository, tablet, capsule, orpacket of powder to be dissolved in a beverage; or each form may beformulated in separate units, e.g, two creams, suppositories, tablets,two capsules, a tablet and a liquid for dissolving the tablet, a packetof powder and a liquid for dissolving the powder, etc.

The term “pharmaceutically acceptable salt” means those salts whichretain the biological effectiveness and properties of the compounds usedin the present invention, and which are not biologically or otherwiseundesirable. For example, a pharmaceutically acceptable salt does notinterfere with the beneficial effect of a compound of the invention ininhibiting MAP kinase and/or HMG-CoA reductase, e.g., in treating aninflamatory, MAP kinase-related and/or HMG-CoA reductase relatedconditon.

Typical salts are those of the inorganic ions, such as, for example,sodium, potassium, calcium, magnesium ions, and the like. Such saltsinclude salts with inorganic or organic acids, such as hydrochloricacid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid,methanesulfonic acid, p-toluenesulfonic acid, acetic acid, fumaric acid,succinic acid, lactic acid, mandelic acid, malic acid, citric acid,tartaric acid or maleic acid. In addition, if the compound(s) contain acarboxy group or other acidic group, it may be converted into apharmaceutically acceptable addition salt with inorganic or organicbases. Examples of suitable bases include sodium hydroxide, potassiumhydroxide, ammonia, cyclohexylamine, dicyclohexyl-amine, ethanolamine,diethanolamine, triethanolamine, and the like.

A pharmaceutically acceptable ester or amide refers to those whichretain biological effectiveness and properties of the compounds used inthe present invention, and which are not biologically or otherwiseundesirable. For example, the ester or amide does not interfere with thebeneficial effect of a compound of the invention in inhibiting MAPkinase and/or HMG-CoA reductase, e.g., in treating an inflamatory, MAPkinase-related and/or HMG-CoA reductase related conditon. Typical estersinclude ethyl, methyl, isobutyl, ethylene glycol, and the like. Typicalamides include unsubstituted amides, alkyl amides, dialkyl amides, andthe like.

In some embodiments, a compound may be administered in combination withone or more other compounds, forms, and/or agents, e.g., as describedabove. Pharmaceutical compositions comprising combinations of a statinlactone with one or more other active agents can be formulated tocomprise certain molar ratios. For example, molar ratios of about 99:1to about 1:99 of statin lactone to the other active agent can be used.Preferably, the range of molar ratios of statin lactone: other activeagent is selected from about 80:20 to about 20:80; about 75:25 to about25:75, about 70:30 to about 30:70, about 66:33 to about 33:66, about60:40 to about 40:60; about 50:50; and about 90:10 to about 10:90. Morepreferably, the molar ratio of statin lactone: other active agent isabout 1:9, and most preferably about 1:1. The two compounds, formsand/or agents may be formulated together, in the same dosage unit e.g.in one cream, suppository, tablet, capsule, or packet of powder to bedissolved in a beverage; or each compound, form, and/or agent may beformulated in separate units, e.g, two creams, suppositories, tablets,two capsules, a tablet and a liquid for dissolving the tablet, a packetof powder and a liquid for dissolving the powder, etc.

If necessary or desirable, the compounds and/or combinations ofcompounds may be administered with still other agents. The choice ofagents that can be co-administered with the compounds and/orcombinations of compounds of the instant invention can depend, at leastin part, on the condition being treated. Agents of particular use in theformulations of the present invention include, for example, any agenthaving a therapeutic effect for kinase-related and/or HMG-CoAreductase-related conditions, including, e.g., drugs used to treatinflammatory conditions. For example, in treatments for acne,formulations of the instant invention may additionally contain one ormore conventional acne treatments, such as keratolytic agents, e.g.,retinoids, particularly retinoic acid; anti-inflammatory agents, such asperoxides, particularly benzoyl peroxide; and antiseborrhoeic agents. Intreamtents for osteoporosis, as another example, formulations mayadditionally contain one or more supplements, such as vitamin D and/orcalcium, and/or one or more biphosphonate medications, e.g., which blockbone resorption.

In still other embodiments, compounds and/or combinations of compoundsdescribed herein can be co-formulated and/or co-administered with agentsuseful for the prevention and/or treatment of atherosclerosis and itssequelae. These agents include, but are not limited to, e.g., inhibitorsof cholesterol ester transferase protein (CETP) (e.g., JTT-705,torcetrapib); inhibitors of sterol acyl-CoA-acyl transferase (ACAT)(e.g., pactimibe, SMP-797, K-604); inhibitors of microsomal triglyceridetransferase protein (MTTP) (e.g., implitipide, JTT-130); modulators ofperoxisome proliferators activated receptors (PPARs) (e.g., binifibrate,gemfibrozil, clinofibrate, ronifibrate, fenofibrate, bezafibrate,LY-929, GW-516, GW-590735, NS-220, LY-674, DRF-10945, SB-641597,AVE-8134, AVE-0847, ciglitazone, pioglitazone, darglitazone,rosiglitazone, isaglitazone, reglitazar, farglitazar, tesaglitazar,balaglitazone, ragaglitazar, rivoglitazone, imiglitazar, edaglitazone,oxeglitazar, muraglitazar); inhibitors of cholesterol absorption (e.g.,ezetimibe, colesevelam hydrochloride, cholestyramine, colestimide,colestipol hydrochloride, BTG-511); vitamins (e.g., niacin); inhibitorsof platelet aggregation (e.g., aspirin, clopidogrel, D-003); inhibitorsof ileal bile acid transport (IBAT) (e.g., S-8921, BARI-1741);inhibitors of lipoprotein-associated phospholipase A2 (Lp-PLA2) (e.g.,SB-480848, SB-659032, SB-677116); inhibitors of squalene synthase (e.g.,TAK-475); antagonists of chemokine CCR2 receptor (e.g., INCB-3284,C-8834, C-1602).

The compound(s) (or pharmaceutically acceptable salts, esters or amidesthereof) may be administered per se or in the form of a pharmaceuticalcomposition wherein the active compound(s) is in an admixture or mixturewith one or more pharmaceutically acceptable carriers. A pharmaceuticalcomposition, as used herein, may be any composition prepared foradministration to a subject. Pharmaceutical compositions for use inaccordance with the present invention may be formulated in conventionalmanner using one or more physiologically acceptable carriers, comprisingexcipients, diluents, and/or auxiliaries, e.g., which facilitateprocessing of the active compounds into preparations that can beadministered. Proper formulation may depend at least in part upon theroute of administration chosen. The compound(s) useful in the presentinvention, or pharmaceutically acceptable salts, esters, or amidesthereof, can be delivered to a patient using a number of routes or modesof administration, including oral, buccal, topical, rectal, transdermal,transmucosal, subcutaneous, intravenous, and intramuscular applications,as well as by inhalation.

For oral administration, the compounds can be formulated readily bycombining the active compound(s) with pharmaceutically acceptablecarriers well known in the art. Such carriers enable the compounds ofthe invention to be formulated as tablets, including chewable tablets,pills, dragees, capsules, lozenges, hard candy, liquids, gels, syrups,slurries, powders, suspensions, elixirs, wafers, and the like, for oralingestion by a patient to be treated. Such formulations can comprisepharmaceutically acceptable carriers including solid diluents orfillers, sterile aqueous media and various non-toxic organic solvents.Generally, the compounds of the invention will be included atconcentration levels ranging from about 0.5%, about 5%, about 10%, about20%, or about 30% to about 50%, about 60%, about 70%, about 80% or about90% by weight of the total composition of oral dosage forms, in anamount sufficient to provide a desired unit of dosage.

Aqueous suspensions for oral use may contain compound(s) of thisinvention with pharmaceutically acceptable excipients, such as asuspending agent (e.g., methyl cellulose), a wetting agent (e.g.,lecithin, lysolecithin and/or a long-chain fatty alcohol), as well ascoloring agents, preservatives, flavoring agents, and the like.

In some embodiments, oils or non-aqueous solvents may be required tobring the compounds into solution, due to, for example, the presence oflarge lipophilic moieties. Alternatively, emulsions, suspensions, orother preparations, for example, liposomal preparations, may be used.With respect to liposomal preparations, any known methods for preparingliposomes for treatment of a condition may be used. See, for example,Bangham et al., J. Mol. Biol. 23: 238–252 (1965) and Szoka et al., Proc.Natl. Acad. Sci. USA 75: 4194–4198 (1978), incorporated herein byreference. Ligands may also be attached to the liposomes to direct thesecompositions to particular sites of action. Compounds of this inventionmay also be integrated into foodstuffs, e.g, cream cheese, butter, saladdressing, or ice cream to facilitate solubilization, administration,and/or compliance in certain patient populations.

Pharmaceutical preparations for oral use can be obtained as a solidexcipient, optionally grinding a resulting mixture, and processing themixture of granules, after adding suitable auxiliaries, if desired, toobtain tablets or dragee cores. Suitable excipients are, in particular,fillers such as sugars, including lactose, sucrose, mannitol, orsorbitol; flavoring elements, cellulose preparations such as, forexample, maize starch, wheat starch, rice starch, potato starch,gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinyl pyrrolidone (PVP). If desired, disintegrating agents may beadded, such as the cross-linked polyvinyl pyrrolidone, agar, or alginicacid or a salt thereof such as sodium alginate. The compounds may alsobe formulated as a sustained release preparation.

Dragee cores can be provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compounds.

Pharmaceutical preparations that can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for administration.

In some preferred embodiments, oral formulations are used to treatAlzheimer's and/or other inflammatory conditons of the central nervoussystem. In some preferred embodiments, oral formulations are used totreat arthritis, rheumatoid arthritis and/or other inflammatoryconditions of the musculoskeletal system. As detailed above, preferredcompositions in such embodiments are those comprising a statin lactoneand a non-statin anti-inflammatory agent.

In some preferred embodiments, oral formulations are used to treatinflammatory conditions of the vascular system especiallyhypercholesterolemia, hyperlipidemia, atherosclerosis, peripheralocclusive disease, myocardial infarction, and stroke. Preferredcompositions in such embodiments are those comprising a statin lactoneand a salt form of a hydroxy acid statin. More preferred arecombinations of atorvastatin lactone with a salt of either atorvastatinor pitavastatin, even more preferably in a molar ratio of about 90:10 toabout 10:90. Additional details of such preferred embodiments for oralformulations are provided in Example 4, as outlined above. Specifically,Example 4b describes a coated tablet formulation comprising atorvastatinlactone and atorvastatin calcium, at a ratio of 1:1. Example 4cdescribes a coated tablet formulation of enteric-coated granulescomprising atorvastatin lactone and pitavastatin calcium, at a ratio of1:1.

For injection, the compounds of the present invention may be formulatedin aqueous solutions, preferably in physiologically compatible bufferssuch as Hank's solution, Ringer's solution, or physiological salinebuffer. Such compositions may also include one or more excipients, forexample, preservatives, solubilizers, fillers, lubricants, stabilizers,albumin, and the like. Methods of formulation are known in the art, forexample, as disclosed in Remington's Pharmaceutical Sciences, latestedition, Mack Publishing Co., Easton P.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation or transcutaneous delivery (forexample subcutaneously or intramuscularly), intramuscular injection oruse of a transdermal patch. Thus, for example, the compounds may beformulated with suitable polymeric or hydrophobic materials (for exampleas an emulsion in an acceptable oil) or ion exchange resins, or assparingly soluble derivatives, for example, as a sparingly soluble salt.

In some embodiments, pharmaceutical compositions comprising one or morecompounds of the present invention exert local and regionalanti-inflammatory effects when administered topically or injected at ornear particular sites of inflammation. Direct topical application, e.g.,of a viscous liquid, gel, jelly, cream, lotion, ointment, suppository,foam, or aerosol spray, may be used for local administration, to producefor example local and/or regional effects. Pharmaceutically appropriatevehicles for such formulation include, for example, lower aliphaticalcohols, polyglycols (e.g., glycerol or polyethylene glycol), esters offatty acids, oils, fats, silicones, and the like. Such preparations mayalso include preservatives (e.g., p-hydroxybenzoic acid esters) and/orantioxidants (e.g., ascorbic acid and tocopherol). See alsoDermatological Formulations: Percutaneous absorption, Barry (Ed.),Marcel Dekker Incl, 1983. In some preferred embodiments, local/topicalformulations comprising a statin lactone and a non-statinanti-inflammatory agent are used to treat inflammatory conditions.Preferred compositions in such embodiments are those comprisingatorvastatin lactone and a non-steroidal anti-inflammatory drug, evenmore preferably in a molar ratio of about 90:10 to about 10:90.

In some preferred embodiments, local/topical formulations are used totreat allergic, inflammatory and/or autoimmune conditions of the skin orskin structures, especially for treating eczema, psoriasis, acne and theeffects of aging. For example, for treating inflammatory and/orautoimmune conditions, a cream comprising a compound of the invention informula I, II, III, or IV may be topically applied to the affected site,for example, sites displaying red plaques or dry scales in psoriasis, orareas of irritation and dryness in dermatitis. Preferred compositions insuch embodiments are those comprising a statin lactone and a salt formof a hydroxy acid statin. More preferred are combinations ofatorvastatin lactone with a salt of either atorvastatin or pitavastatin,even more preferably in a molar ratio of about 90:10 to about 10:90.

Pharmaceutical compositions of the present invention may contain acosmetically or dermatologically acceptable carrier. Such carriers arecompatible with skin, nails, mucous membranes, tissues and/or hair, andcan include any conventionally used cosmetic or dermatological carriermeeting these requirements. Such carriers can be readily selected by oneof ordinary skill in the art. In formulating skin ointments, a compoundor combination of compounds of the instant invention may be formulatedin an oleaginous hydrocarbon base, an anhydrous absorption base, awater-in-oil absorption base, an oil-in-water water-removable baseand/or a water-soluble base.

The compositions according to the present invention may be in any formsuitable for topical application, including aqueous, aqueous-alcoholicor oily solutions, lotion or serum dispersions, aqueous, anhydrous oroily gels, emulsions obtained by dispersion of a fatty phase in anaqueous phase (O/W or oil in water) or, conversely, (W/O or water inoil), microemulsions or alternatively microcapsules, microparticles orlipid vesicle dispersions of ionic and/or nonionic type. Thesecompositions can be prepared according to conventional methods. Otherthan the compounds of the invention, the amounts of the variousconstituents of the compositions according to the invention are thoseconventionally used in the art. These compositions in particularconstitute protection, treatment or care creams, milks, lotions, gels orfoams for the face, for the hands, for the body and/or for the mucousmembranes, or for cleansing the skin. The compositions may also consistof solid preparations constituting soaps or cleansing bars.

Compositions of the present invention may also contain adjuvants commonto the cosmetic and dermatological fields, such as hydrophilic orlipophilic gelling agents, hydrophilic or lipophilic active agents,preserving agents, antioxidants, solvents, fragrances, fillers,sunscreens, odor-absorbers and dyestuffs. The amounts of these variousadjuvants are those conventionally used in the fields considered and,for example, are from about 0.01% to about 20% of the total weight ofthe composition. Depending on their nature, these adjuvants may beintroduced into the fatty phase, into the aqueous phase and/or into thelipid vesicles.

Preferred embodiments of ointment formulations are described in Example4. Example 4a describes a gel formulation comprising atorvastatinlactone and atorvastatin calcium. Example 4d describes a hydrophilicointment comprising atorvastatin lactone and naproxen sodium. Example 4edescribes a polyethylene glycol ointment comprising atorvastatin lactoneand diclofenac. Example 4g describes an ointment comprising atorvastatinlactone and indomethacin. Example 4h describes a gel formulationincluding atorvastatin lactone and indomethacin. Example 4i describes acream formulation comprising atorvastatin lactone and indomethacin.Example 4j describes a skin ointment comprising atorvastatin lactone andpetrolatum USP. Example 4k describes a skin ointment comprisingfluvastatin lactone and white petrolatum. Example 4l describes a skinointment comprising cerivastatin lactone. Example 4m describes a skinointment comprising pitavastatin lactone and polyethylene glycol. Inmore preferred embodiments, the ointment, gel, and/or cream formulationsdescribed in Examples 4g, 4h, and 4i, respectively, provide synergisticinhibitory effects in treating inflammation, e.g., as detailed inExample 8 below.

In some embodiments, ocular allergic, inflammatory and/or autoimmuneconditions can be effectively treated with ophthalmic solutions,suspensions, ointments or inserts comprising a compound or combinationof compounds of the present invention. Preferred embodiments offormulations for ocular use are provided in Example 4. Specifically,Example 4n describes an isotonic solution comprising rosuvastatin;Example 4o describes an ointment comprising cerivastatin lactone; andExample 4p describes an ointment comprising atorvastatin lactone, eachof which can be used for ocular application.

In some embodiments, allergic, inflammatory and/or autoimmune conditionsof the ear can be effectively treated with otic solutions, suspensions,ointments or inserts comprising a compound or combination of compoundsof the present invention. Preferred embodiments of formulations for oticuse are provided in Example 4. Specifically, Example 4q describes asolution comprising fluvastatin sodium and glycerin for otic use.

In some preferred embodiments, the compounds of the present inventionare delivered in soluble rather than suspension form, which allows formore rapid and quantitative absorption to the sites of action. Ingeneral, formulations such as jellies, creams, lotions, suppositoriesand ointments can provide an area with more extended exposure to thecompounds of the present invention, while formulations in solution,e.g., sprays, provide more immediate, short-term exposure.

In some embodiments relating to topical/local application, thepharmaceutical compositions can include one or more penetrationenhancers. For example, the formulations may comprise suitable solid orgel phase carriers or excipients that increase penetration or helpdelivery of compounds or combinations of compounds of the inventionacross a permeability barrier, e.g., the skin. Many of thesepenetration-enhancing compounds are known in the art of topicalformulation, and include, e.g., water, alcohols (e.g., terpenes likemethanol, ethanol, 2-propanol), sulfoxides (e.g., dimethyl sulfoxide,decylmethyl sulfoxide, tetradecylmethyl sulfoxide), pyrrolidones (e.g.,2-pyrrolidone, N-methyl-2-pyrrolidone, N-(2-hydroxyethyl)pyrrolidone),laurocapram, acetone, dimethylacetamide, dimethylformamide,tetrahydrofurfuryl alcohol, L-α-amino acids, anionic, cationic,amphoteric or nonionic surfactants (e.g., isopropyl myristate and sodiumlauryl sulfate), fatty acids, fatty alcohols (e.g., oleic acid), amines,amides, clofibric acid amides, hexamethylene lauramide, proteolyticenzymes, α-bisabolol, d-limonene, urea and N,N-diethyl-m-toluamide, andthe like Additional examples include humectants (e.g., urea), glycols(e.g., propylene glycol and polyethylene glycol), glycerol monolaurate,alkanes, alkanols, ORGELASE, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and/or otherpolymers. In some embodiments, the pharmaceutical compositions willinclude one or more such penetration enhancers.

In some embodiments, the pharmaceutical compositions for local/topicalapplication can include one or more antimicrobial preservatives such asquaternary ammonium compounds, organic mercurials, p-hydroxy benzoates,aromatic alcohols, chlorobutanol, and the like.

Gastrointestinal allergic, inflammatory and/or autoimmune conditions canbe effectively treated with orally- or rectally delivered solutions,suspensions, ointments, enemas and/or suppositories comprising acompound or combination of compounds of the present invention.Local/topical formulations are preferred for therapy of Crohn's colitisand other allergic, inflammatory and/or autoimmune diseases of thegastrointestinal system.

In treating inflammatory bowel disease, for example, a suppositoryformulation of a compound or combination of compounds disclosed hereincan be used. In such embodiments, the active ingredient can produce abenefit locally at or near the site of application, rather thansystemically, by inhibiting a MAP kinase inhibitor, e.g., p38α MAPkinase. In some preferred embodiments, a lactone form (formula I) of aknown statin is used in formulations for topical inhibition of MAPkinase. In more preferred embodiments, the statin lactone used topicallyis a synthetic statin lactone, such as atorvastatin, cerivastatin,fluvastatin, pitavastatin, glenvastatin, and/or rosuvastatin, including,for example, structures provided in FIGS. 7 and 8. In some preferredembodiments, compounds modified to favor a closed ring structure, suchas formulas III and IV of FIG. 9, are used in formulations for topicalinhibition of MAP kinase. In more preferred embodiments, the modifiedcompound is derived from a synthetic statin lactone, such asatorvastatin, cerivastatin, fluvastatin, pitavastatin, glenvastatin,and/or rosuvastatin.

Details of preferred embodiments for enema and suppository formulationsare described in Example 4. Specifically, Example 4r describes aretention enema comprising cerivastatin sodium; Example 4s describes aretention enema comprising pitavastatin lactone; Example 4t describes arectal suppository comprising fluvastatin lactone; Example 4u describesa rectal suppository comprising atorvastatin lactone; and Example 4fdescribes a rectal suppository comprising atorvastatin lactone andaminosalicylic acid.

Respiratory allergic, inflammatory and/or autoimmune conditions can beeffectively treated with aerosol solutions, suspensions or dry powderscomprising a compound or combination of compounds of the presentinvention. Administration by inhalation is particularly useful intreating inflammatory conditions of the lung. The aerosol can beadministered through the respiratory system or nasal passages. Forexample, one skilled in the art will recognize that a composition of thepresent invention can be suspended or dissolved in an appropriatecarrier, e.g., a pharmaceutically acceptable propellant, andadministered directly into the lungs using a nasal spray or inhalant.For example, an aerosol formulation comprising a MAP kinase and/or HMGCoA reductase inhibitor can be dissolved, suspended or emulsified in apropellant or a mixture of solvent and propellant, e.g., foradministration as a nasal spray or inhalant. Aerosol formulations maycontain any acceptable propellant under pressure, preferably acosmetically or dermatologically or pharmaceutically acceptablepropellant, as conventionally used in the art.

An aerosol formulation for nasal administration is generally an aqueoussolution designed to be administered to the nasal passages in drops orsprays. Nasal solutions can be similar to nasal secretions in that theyare generally isotonic and slightly buffered to maintain a pH of about5.5 to about 6.5, although pH values outside of this range canadditionally be used. Antimicrobial agents or preservatives can also beincluded in the formulation.

An aerosol formulation for inhalations and inhalants can be designed sothat the compound or combination of compounds of the present inventionis carried into the respiratory tree of the subject when administered bythe nasal or oral respiratory route. Inhalation solutions can beadministered, for example, by a nebulizer. Inhalations or insufflations,comprising finely powdered or liquid drugs, can be delivered to therespiratory system as a pharmaceutical aerosol of a solution orsuspension of the compound or combination of compounds in a propellant,e.g., to aid in disbursement. Propellants can be liquefied gases,including halocarbons, for example, fluorocarbons such as fluorinatedchlorinated hydrocarbons, hydrochlorofluorocarbons, andhydrochlorocarbons, as well as hydrocarbons and hydrocarbon ethers.

Halocarbon propellants useful in the present invention includefluorocarbon propellants in which all hydrogens are replaced withfluorine, chlorofluorocarbon propellants in which all hydrogens arereplaced with chlorine and at least one fluorine, hydrogen-containingfluorocarbon propellants, and hydrogen-containing chlorofluorocarbonpropellants. Halocarbon propellants are described in Johnson, U.S. Pat.No. 5,376,359, issued Dec. 27, 1994; Byron et al., U.S. Pat. No.5,190,029, issued Mar. 2, 1993; and Purewal et al., U.S. Pat. No.5,776,434, issued Jul. 7, 1998. Hydrocarbon propellants useful in theinvention include, for example, propane, isobutane, n-butane, pentane,isopentane and neopentane. A blend of hydrocarbons can also be used as apropellant. Ether propellants include, for example, dimethyl ether aswellas the ethers. An aerosol formulation of the invention can alsocomprise more than one propellant. For example, the aerosol formulationcan comprise more than one propellant from the same class, such as twoor more fluorocarbons; or more than one, more than two, more than threepropellants from different classes, such as a fluorohydrocarbon and ahydrocarbon. Pharmaceutical compositions of the present invention canalso be dispensed with a compressed gas, e.g., an inert gas such ascarbon dioxide, nitrous oxide or nitrogen.

Aerosol formulations can also include other components, for example,ethanol, isopropanol, propylene glycol, as well as surfactants or othercomponents such as oils and detergents. These components can serve tostabilize the formulation and/or lubricate valve components.

The aerosol formulation can be packaged under pressure and can beformulated as an aerosol using solutions, suspensions, emulsions,powders and semisolid preparations. For example, a solution aerosolformulation can comprise a solution of a compound of the invention suchas a novel HMG-CoA reductase inhibitor in (substantially) purepropellant or as a mixture of propellant and solvent. The solvent can beused to dissolve the compound and/or retard the evaporation of thepropellant. Solvents useful in the invention include, for example,water, ethanol and glycols. Any combination of suitable solvents can beuse, optionally combined with preservatives, antioxidants, and/or otheraerosol components.

An aerosol formulation can also be a dispersion or suspension. Asuspension aerosol formulation may comprise a suspension of a compoundor combination of compounds of the instant invention, e.g., an HMG CoAreductase inhibitor, and a dispersing agent. Dispersing agents useful inthe invention include, for example, sorbitan trioleate, oleyl alcohol,oleic acid, lecithin and corn oil. A suspension aerosol formulation canalso include lubricants, preservatives, antioxidant, and/or otheraerosol components.

An aerosol formulation can similarly be formulated as an emulsion. Anemulsion aerosol formulation can include, for example, an alcohol suchas ethanol, a surfactant, water and a propellant, as well as a compoundor combination of compounds of the invention, e.g., an HMG-CoA reductaseinhibitor. The surfactant used can be nonionic, anionic or cationic. Oneexample of an emulsion aerosol formulation comprises, for example,ethanol, surfactant, water and propellant. Another example of anemulsion aerosol forumalation comprises, for example, vegetable oil,glyceryl monostearate and propane.

Preferred embodiments of aerosol formulations are described in Example4. Specifically, Example 4v describes a dry powder aerosol formulationcomprising pitavastatin lactone and lactose. Example 4w describes a drypowder aerosol formulation comprising fluvastatin sodium. Example 4xdescribes a dry powder aerosol formulation comprising atorvastatinlactone. Example 4y describes a metered-dose aerosol formulationcomprising atorvastatin lactone.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions wherein the active ingredients are present in aneffective amount, i.e., in an amount effective to achieve therapeuticand/or prophylactic benefit in at least one of a MAP kinase-relatedcondition and an HMG-CoA reductase-related condition. The actual amounteffective for a particular application will depend on the condition orconditions being treated, the condition of the subject, the formulation,and the route of administration, as well as other factors known to thoseof skill in the art. Determination of an effective amount of a MAPkinase and/or HMG-CoA reductase inhibitor is well within thecapabilities of those skilled in the art, in light of the disclosureherein, and will be determined using routine optimization techniques.

The effective amount for use in humans can be determined from animalmodels. For example, a dose for humans can be formulated to achievecirculating, liver, topical and/or gastrointestinal concentrations thathave been found to be effective in animals. One skilled in the art candetermine the effective amount for human use, especially in light of theanimal model experimental data described herein. Example 8, providedbelow, details an experiment where an inflammatory condition was inducedin mice, compositions of the present invention were administered, andanti-inflammatory effect obsereved. As described in more detail below,Table II shows the resulting percentage of inhibition and effectiveamounts of compounds and combinations of compounds of the instantinvention. Based on this animal data, and other types of similar data,those skilled in the art can determine the effective amounts ofcompositions of the present invention appropriate for humans.

The effective amount when referring to a compound or combination ofcompounds of the invention will generally mean the dose ranges, modes ofadministration, formulations, etc., that have been recommended orapproved by any of the various regulatory or advisory organizations inthe medical or pharmaceutical arts (e.g., FDA, AMA) or by themanufacturer or supplier. Effective amounts of HMG-CoA reductaseinhibitors can be found, for example, in the Physicians Desk Reference.For example, daily doses for atorvastatin calcium range from about 2 mgto about 50 mg, from about 3 mg to about 30 mg, typically about 10 mg. Adaily dose for cerivastatin sodium is about 200 μg, while daily dosesfor fluvastatin sodium, rosuvastatin sodium, pravastatin sodium andsimvastatin are each about 20 mg. Some preferred compounds of thisinvention, e.g., analogs of HMG-CoA reductase inhibitors, may be usefulin about the same dosages, or less than or more than dosages typical ofknown HMG-CoA reductase inhibitors.

Effective amounts of MAP kinase inhibitors can be found, for example, inpublished reports of the results of human clinical trials. Generally,the recommended dosage for a MAP kinase inhibitor of the presentinvention, e.g., a p38α MAP kinase inhibitor, is a dose of about 0.01mg/kg to about 1,000 mg/kg, more preferably from about 0.1 mg/kg toabout 20 mg/kg on a daily basis, provided orally. The inhibitor istypically administered in a dose of about 100 mg, which is in the rangeof doses that will be useful in the present invention. Using otherroutes of administration, it is believed that a dose of about 0.01mg/kg/day to about 1,000 mg/kg/day of a MAP kinase inhibitor will beused; preferably a dose between about 0.1 mg/kg/day and about 20mg/kg/day will be used.

Generally, the recommended dosage for an HMG-CoA reductase inhibitor ofthe present invention is a dose of about 0.01 mg/kg to about 1,000mg/kg, more preferably from about 0.1 mg/kg to about 20 mg/kg on a dailybasis, provided orally. The inhibitor is typically administered in adose of about 10 mg, which is in the range of doses that will be usefulin the present invention. Using other routes of administration, it isbelieved that a dose of about 0.01 mg/kg/day to about 1,000 mg/kg/day ofan HMG-CoA reductase inhibitor will be used; preferably a dose betweenabout 0.1 mg/kg/day and about 1 mg/kg/day will be used.

Further, appropriate doses for a statin lactone, hydroxy acid form of astatin or non-statin anti-inflammatory agent can be determined based onin vitro experimental results provided herein. For example, the in vitropotency of a compound in inhibiting HMG-CoA reductase and/or inhibitinginflammation mediators provides information useful in the development ofeffective in vivo dosages to achieve similar biological effects.

Effective amounts of compounds and/or combinations of compounds of theinstant invention for use in increasing HDL levels can similarly bedetermined based on in vitro experimental data, including animal modeldata. For example, an animal model can be used to determine a percentageincrease in HDL levels that would be desirable in humans, andcorresponding effective doses of the compound(s) or combinations ofcompounds to achive such levels.

In some embodiments, administration of compounds of the presentinvention may be intermittent, for example administration once every twodays, every three days, every five days, once a week, once or twice amonth, and the like. In some embodiments, the amount, forms, and/oramounts of the different forms may be varied at different times ofadministration. For example, at one point in time, the acid form of acompound of the present invention may be administered, while at anothertime the corresponding lactone form may be used.

A person of skill in the art would be able to monitor in a patient theeffect of administration of a particular compound. For example,cholesterol levels can be determined by measuring LDL, HDL, and/or totalserum cholesterol levels. The release of pro-inflammatory cytokines canbe determined by measuring TNF-αand/or IL-1β. Other techniques would beapparent to one of skill in the art.

IV. Rational Design of Kinase and/or HMG-CoA Reductase Inhibitors

Still another aspect of the present invention relates to methods ofobtaining and/or making a composition for inhibiting a MAP kinase and/orHMG CoA reductase by designing a compound of formula I/II/III/IV;testing whether the compound inhibits a MAP kinase and/or HMG CoAreductase; and using the compound in making a composition for inhibitinga MAP kinase and/or HMG-CoA reductase. More preferably, the inventionrelates to methods for designing and testing compounds of formula I/IIthat are capable of inhibiting both MAP kinase and HMG-CoA reductase.

By “formula I/II” it is meant that either the δ-lactone or the hydroxycarboxylic acid forms, or both forms, may be responsible for inhibitionof a MAP kinase, HMG-CoA reductase or both. FIG. 11 illustrates a designapproach for developing compounds that inhibit a MAP kinase and/orHMG-CoA reductase.

FIG. 11 a illustrates an approach in which known inhibitors of MAPkinases are systematically varied and tested for HMG-CoA reductaseinhibitory activity. In this approach, known inhibitors of MAP kinasesand analogs thereof are appended or substituted with an A-δ-lactone oran A-hydroxy carboxylic acid group of formulas I or II, respectively.Thus, in some embodiments, the lipophilic moiety (X) is a known MAPkinase inhibitor or a lipophilic moiety thereof. For example, FIG. 4illustrates known p38α MAP kinase inhibitors that may be used indesigning some preferred embodiments. In other embodiments, thelipophilic moiety is an analog of a MAP kinase inhibitor, for example,selected on the basis of structural diversity or similarity to anHMG-CoA reductase inhibitor, preferably to a lipophilic moiety of anHMG-CoA reductase inhibitor, or on the basis of structural compatibilitywith binding to HMG-CoA reductase, for example, using pharmacophoremodeling to indicate binding compatibility. For example, FIG. 5illustrates MAP kinase analogs preferred in some embodiments.

FIG. 11 b illustrates an approach in which known inhibitors of HMG-CoAreductase are systematically varied and tested for MAP kinase inhibitoryactivity. In this approach, lipophilic moieties (X) of known inhibitorsof HMG-CoA reductase are systematically varied, resulting in analogs.FIG. 6, for example, illustrates known statins that can be used indesigning some preferred embodiments. In some embodiments, thelipophilic moiety analog is selected on the basis of structuraldiversity or similarity to a MAP kinase inhibitor, preferably to alipophilic moiety of a MAP kinase inhibitor, on the basis of structuralcompatibility with binding to a MAP kinase, such as p38α MAP kinase, forexample, using pharmacophore modeling to indicate binding compatibility.For example, FIG. 7 illustrates preferred lipophilic moiety analogs, andFIG. 8 illustrates specific examples of statin analogs, which are evenmore preferred in some embodiments.

The rational design methods of the present invention are aided by thecurrent understanding of the crystal structures of HMG-CoA reductase andMAP kinases. The X-ray structure of p38α MAP kinase, for example, hasbeen shown to comprise an N-terminal domain with an ATP binding pocket,and a C-terminal domain with a catalytic site, metal binding site, andphophorylation lip. The two domains are connected by a hinge region, towhich the substrate binds. Further, a direct correlation has been shownbetween the “tightness” of binding of a candidate compound to the enzymeand the in vitro cellular activity of the compound.

FIG. 11 c illustrates an approach in which the lipophilic moiety ofcompounds of formula I or II is varied and tested for MAP kinase and/orHMG-CoA reductase inhibitory activity. In some embodiments, thelipophilic moiety is randomly selected. In some embodiments, thelipophilic moiety is selected on the basis of structural diversity orsimilarity to a MAP kinase inhibitor or on the basis of structuralcompatibility with binding to a MAP kinase, for example, usingpharmacophore modeling to indicate binding compatibility. In someembodiments, the lipophilic moiety is selected on the basis ofstructural diversity or similarity to an HMG-CoA reductase inhibitor oron the basis of structural compatibility with binding to an HMG-CoAreductase, for example, using pharmacophore modeling to indicate bindingcompatibility. Selected lipophilic moieties can be appended with anA-δ-lactone or an A-hydroxy carboxylic acid group of formulas I or II,respectively, and then tested for inhibition of a MAP kinase and/orinhibition of an HMG-CoA reductase.

Compounds can be designed and tested entirely using computationalmethods or a portion of such designing and testing can be donecomputationally and the remainder done with wet lab techniques.

Testing involves evaluation of the designed compounds for inhibitoryactivity towards a MAP kinase and/or HMG-CoA reductase. In someembodiments, the collection of designed analogs may be evaluated bycomputational methods to predict their activity in inhibiting a MAPkinase and/or HMG-CoA reductase, without physically synthesizing thecompounds. Such computational methods may also be used to predict otherproperties of the compounds, such as solubility, membrane penetrability,metabolism and toxicity.

In some embodiments, testing involves synthesizing the designedcompounds and evaluating their activity in inhibiting a MAP kinaseand/or HMG-CoA reductase in one or more biological assays via wet labtechniques. Known methods for the synthesis of inhibitors of HMG-CoAreductase and MAP kinases can be adapted to prepare the designed analogsin either the δ-lactone or the hydroxy carboxylic acid form, as well asin carboxylate (salt) form.

The activity of the synthesized compound can then be evaluated by abiological assay, which directly or indirectly reflects the inhibitionof a MAP kinase, and/or the inhibition of HMG-CoA reductase.Representative biological assays include, but are not limited to, 1)cell-free studies of MAP kinase inhibition; 2) cell-free studies ofHMG-CoA reductase inhibition; 3) whole-cell studies of inhibition ofinflammatory responses (such as cytokine production and/or release uponchallenge by agents, including lipopolysaccharide (LPS)); 4) whole cellstudies of terpene and sterol biosynthesis; 5) in vivo models ofefficacy against MAP kinase-related conditions, such as inflammatoryand/or autoimmune conditions, including arthritis, rheumatoid arthritis,osteoarthritis, vascular inflammatory conditions, inflammatory boweldisease, psoriasis, topical dermatitis, eczema, endotoxemia, sepsis, andtoxic shock syndrome, as well as transplant rejection; 6) in vivo modelsof efficacy in treating HMG-CoA reductase-related conditions, such ashypercholesterolemia, lipid disorders such as hyperlipidemia, andatherogenesis and its sequelae, including atherosclerosis, othervascular inflammatory conditions, myocardial infarction, ischemicstroke, occlusive stroke, peripheral occlusive disease, and otherperipheral vascular diseases.

With respect to in vitro assays, the ability of a candidate compound toinhibit MAP kinase and/or HMG-CoA reductase activity can be evaluated bycontacting the compound with an assay mixture for measuring activity ofa MAP kinase and/or HMG-CoA reductase, and determining the activity ofthe enzyme in the presence and absence of the compound. A decrease inactivity of a MAP kinase in the presence as opposed to the absence ofthe compound indicates a MAP kinase inhibitor. A decrease in theactivity of HMG-CoA reductase in the presence as opposed to the absenceof the compound indicates an HMG-CoA reductase inhibitor. Both MAPkinase and HMG-CoA reductase are known and commercially available,facilitating simple in vitro assays for inhibitory activity.

An example of a cell-free MAP kinase assay involves that described inClerk and Sugden, FEBS Letters, 426:93–96 (1998), incorporated herein byreference. Briefly, serum can be withdrawn from neonatal rats andmyocytes exposed to sorbitol (about 30 min) in the absence or presenceof about 10 μM or less of a candidate compound. SAPKs/JNKs can beseparated by FPLC on a Mono Q HR5/5 column where the MAP kinases areeluted using about a 30 ml linear NaCl gradient (about 0 to about 0.5 MNaCl). They can be assayed by the direct method with myelin basicprotein (MBP) or about 0.5 mg/ml glutathioneS-transferase-GST-c-Jun(1-135) as substrate, where the assay mixcontains about 0.1% (v/v) dimethyl sulphoxide or about 10 μM of acandidate compound (final concentrations). Samples of fractions can betaken for in-gel kinase assays. Fractions may be pooled and concentratedby ultra-filtration and prepared for immunoblot analysis. For MAP-KAPK2,proteins can be applied to a Mono S HR5/5 column and MAP-KAPK2 purifiedand assayed. GST-c-Jun(1-135) can be used to “pull down” totalSAPKs/JNKs from myocyte extracts. Pellets can be washed in kinase assaybuffer (for example, about 20 mM HEPES pH about 7.7, about 2.5 mM MgCl₂,about 0.1 mM EDTA, about 20 mM β-glycerophosphate) containing the finalconcentrations of a candidate compound. The pellets can be re-suspendedin about 15 μl kinase assay buffer containing twice the finalconcentrations of a candidate compound and phosphorylation can beinitiated with about 15 μl of kinase assay buffer containing about 10 μMATP and about 1 μCi [γ-³²P]ATP. JNK1 isoforms can be immunoprecipitatedfrom myocyte extracts using antibodies. The pellets can be washed inkinase assay buffer containing the final concentrations of a candidatecompound. GST-c-Jun(1-135) in about 15 μl kinase assay buffer containingtwice the final concentrations of a candidate compound can be added andphosphorylation initiated with about 15 μl of kinase assay buffercontaining about 20 μM ATP and about 2 μCi [γ-³²P]ATP. Example 5, below,provides further details of a human p38α MAP kinase inhibition assay, asresults using a number of candidate compounds.

An example of a cell-free HMG-CoA reductase assay involves radiometricprocedures described in Shum et al., Ther. Drug Monit. 20:41–49 (1998),incorporated herein by reference. Briefly, about 150 μg/mL of HMG-CoAreductase can be incubated with a candidate compound, together withabout 12 μM [¹⁴C]HMG-CoA and about 200 μM NADPH in about 200 μL 0.2Mphosphate buffer (pH about 7.2) for about 0.5 h at about 37° C. The[¹⁴C]mevalonate that forms can be converted under acidic conditions to[¹⁴C]mevalonolactone and separated from un-reacted substrate, forexample, by ion-exchange chromatography, and then quantified, forexample, by liquid scintillation counting.

An example of a whole cell assay of inhibition of inflammatory responsesinvolves evaluating murine thymic T cell proliferation and IL-2production or gene expression in the presence and absence of a candidatecompound. Methods for measuring T cell proliferation and IL-2 productionare standard, well known techniques in the art. Other examples of wholecell assays for inflammation are also known in the art, for example, asdescribed in Welker et al., Int. Arch. Allergy & Immunology, 109:110–115(1996); Suhindler et al., Blood, 75:40 (1990); and Gulenboik et al.,JBL, 266:19490 (1991), incorporated herein by reference. Example 6,provided below, further details a whole-cell anti-inflammation assayuseful in certain rational design embodiments of the present invention.Example 7, provided below, further details a whole-cell LPS-stimulatedTNF-α release assay also useful in certain rational design embodimentsof the present invention.

Animal models used to reflect inflammatory or immune responses can beutilized to evaluate MAP kinase inhibitory activity in vivo. Exemplaryanimal models include, but are not limited to, release of inflammatorymediators in response to LPS administration to mice or rats; the mouseacute irritant model; inbred NC/Nga mice, which develop chronicrelapsing skin inflammation when reared under non-pathogen-freeconditions; Balb/c mice, which develop dermatitis when injected withShistosoma japonica glutathione-S-transferase; mice sensitized byrepetitive epicutaneous exposure to ovalbumin antigen that model atopicdermatitis; and dextran sulfate sodium, trinitrobenzene sulfonic acid,and oxazolone-induced colitis, which model inflammatory bowel disease.See also, Nagai et al., J. Pharmacol. Exp. Therapeutics 288:43–50(1999); Boismenu et al., J. Leukoc. Biol., 67:267–278 (2000); andBlumberg et al., Curr. Opin. Immunol. 11:648–656 (1999). Further,Example 8 below provides more details of a topical inflammation animalmodel useful in certain rational design embodiments of the presentinvention.

In some preferred embodiments, the activity or potency of a compound offormula I/II is similar towards a MAP kinase and HMG-CoA reductase,preferably as measured by whole cell and/or in vivo assays of IC50 orED50 values, as described in more detail above. In preferredembodiments, potencies with respect to a MAP kinase and HMG-CoAreductase differ by no more than a factor of about 1000. Morepreferably, potencies differ by no more than a factor of about 100. Mostpreferably, potencies differ by no more than a factor of about 10. In ahighly preferred embodiment, the lactone form of a compound (formula I)is the more potent form against a MAP kinase, the hydroxy carboxylicacid form or carboxylate (salt) form (formula II) is the more potentform against HMG-CoA reductase, and the potencies of these forms againsttheir respective targets differs by no more than a factor of about 10.

In other preferred embodiments, the potency of a compound of formulaI/II against a MAP kinase is greater than its potency against HMG-CoAreductase. In such embodiments, potencies with respect to a MAP kinaseand HMG-CoA reductase differ by at least a factor of about 10. Morepreferably, potencies differ by more than a factor of about 100. Mostpreferably, potencies differ by more than a factor of about 1000.

In yet other preferred embodiments, the potency of a compound of formulaI/II against HMG-CoA reductase is greater than its potency against a MAPkinase. In such embodiments, potencies with respect to HMG-CoA reductaseand a MAP kinase differ by at least a factor of about 10. Morepreferably, potencies differ by more than a factor of about 100. Mostpreferably, potencies differ by more than a factor of about 1000.

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

EXAMPLES

The following examples are intended to illustrate details of theinvention, without thereby limiting it in any manner.

Example 1

Synthesis of Atorvastatin Lactone.

5.0 g (8.6 mmole) of atorvastatin calcium was dissolved in 300 mL ethylacetate and washed with 300 mL 10% (w/v) aqueous sodium hydrogen sulfatesolution (pH 3). The organic phase was dried over anhydrous magnesiumsulfate, filtered and the solvent removed under reduced pressure toafford 2.85 g (5.11 mmole) of atorvastatin acid. This material wasdissolved in 300 mL anhydrous toluene and heated at 60° C. for 40 hours,at which time analytical thin-layer chromatography using 4:1 methylenechloride:acetone eluent indicated near-complete conversion of thestarting acid to a less polar product. The toluene was removed underreduced pressure and the reaction mixture was fractionated on 300 cc ofsilica gel using 4:1 methylene chloride: acetone eluent to afford, aftercombining, concentrating and drying of the appropriate fractions, 2.14 g(3.96 mmol, 46% overall) of atorvastatin lactone as a white foam. The400 MHz ¹H nuclear magnetic resonance (NMR) spectrum and theelectrospray mass spectrum (ES-MS) were consistent with the lactoneproduct. ¹H NMR (Me₂SO-d₆) δ 9.80 (s, 1H), 7.49 (d, 2H), 7.25–7.15 (m,6H), 7.05 (s, 4H), 6.99 (t, 2H), 5.15 (d, 1H), 4.46 (br s, 1H), 4.02 (s,1H), 3.97 (m, 1H), 3.89 (m, 1H), 3.21 (q, 1H), 2.55 (dd, 1H), 2.32 (dd,1H), 1.74 (br s, 2H), 1.6 (m, 2H), 1.36 (d, 6H). ES-MS: obsvd. m/z 541([MH]⁺).

Example 2

Synthesis of Fluvastatin Lactone.

7.0 g (16 mmole) of fluvastatin sodium was dissolved in 300 mL ethylacetate and washed with 300 mL 10% (w/v) aqueous sodium hydrogen sulfatesolution (pH 3). The organic phase was dried over anhydrous magnesiumsulfate, filtered and the solvent removed under reduced pressure toafford 5.76 g (14.0 mmole) of fluvastatin acid. This material wasdissolved in 300 mL anhydrous toluene and stirred at room temperaturefor 7 days, at which time analytical thin-layer chromatography using 5:1methylene chloride:acetone eluent indicated approximately 30% conversionof the starting acid to a less polar product. The toluene was removedunder reduced pressure and the reaction mixture was fractionated on 400cc of silica gel using 5:1 methylene chloride: acetone eluent to afford,after combining, concentrating and drying of the appropriate fractions,2.02 g (5.14 mmol, 32% overall) of fluvastatin lactone as a white foam.The 400 MHz ¹H nuclear magnetic resonance (NMR) spectrum and theelectrospray mass spectrum (ES-MS) were consistent with the lactoneproduct. ¹H NMR (Me₂SO-d₆) δ 7.7 (d, 1H), 7.4 (br s, 3H), 7.3 (m, 2H),7.2 (t, 1H), 7.1 (t, 1H) 6.8 (t, 1H), 5.7 (dd, 1H), 5.3 (s, 1H), 5.2 (m,1H), 4.9 (m, 1H), 4.1 (br s, 1H), 2.7 (dd, 1H), 2.4 (d, 1H), 1.8 (d,1H), 1.7 (t, 1H), 1.57 (d, 6H). ES-MS: obsvd. m/z 394 ([MH]⁺).

Also obtained was a slightly more polar product which ¹H NMR indicatedto be the threo-epimer of fluvastatin lactone formed by inversion at theC5 lactone ester center, in accord with the findings of Stokker andPitzenberger (Heterocycles 1987, 26, 157). This isomer was obtained inan amount of 0.173 g (0.440 mmole, 2.8% overall). ¹H NMR (Me₂SO-d₆) δ7.7 (d, 1H), 7.42 (m, 3H), 7.3 (t, 2H), 7.2 (t, 1H), 7.05 (t, 1H), 6.8(t, 1H), 5.7 (dd, 1H), 5.2 (s, 1H), 4.9 (m, 1H), 4.9 (m, 2H), 4.1 (m,1H), 2.8 (dd, 1H), 2.7 (dd, 1H), 2.3 (dd, 1H), 2.2 (m, 1H), 1.6 (d, 6H).

Using the procedures outlined in Examples 1 and 2, other compounds ofFormula IIa/IIb are converted to compounds of Formula I.

Example 3

Synthesis of Simvastatin Sodium.

1.43 g (3.42 mmole) of simvastatin lactone was dissolved in 10 mLacetonitrile and treated with water (5 mL) and sodium hydroxide (151 mg,3.78 mmole). The reaction was stirred at room temperature for 3 days, atwhich time analytical thin-layer chromatography using 4:1 methylenechloride:acetone eluent indicated essentially complete conversion of thestarting lactone to a more polar product. The reaction mixture was thendiluted to 50 nL with 1:1 acetonitrile:water, frozen and lyophilized toafford 1.22 g (2.66 mmole, 77.8%) of simvastatin sodium as a fluffywhite solid. The 400 MHz ¹H nuclear magnetic resonance (NMR) spectrumwas consistent with the sodium carboxylate product. ¹H NMR (Me₂SO-d₆) δ7.6 (br s, 1H), 5.95 (d, 1H), 5.8 (m, 1H), 5.5 (s, 1H), 5.1 (s, 1H), 4.6(s, 1H), 3.7 (br s, 1H), 3.5 (br s, 1H), 2.3 (m, 2H), 2.2 (d, 1H), 2.0(m, 2H), 1.8 (m, 2H), 1.6–1.3 (5H), 1.2 (br s, 3H), 1.0 (9H), 0.80 (m,3H), 0.75 (m, 3H).

Using the procedure outlined in Example 3, other compounds of Formula Iare converted to compounds of Formula IIb.

Example 4

Pharmaceutical Compositions Comprising a Compound(s) of Formula I/IIa/Ibor III or IV, optionally with a Known Anti-Inflammatory Agent(s), forLocal/Regional Applications.

Example 4a

Gel Formulation

Atorvastatin lactone  0.1 g Atorvastatin calcium  0.1 g Polyglycerylacrylate (Norgel)   3 g Polyacrylamide/C13–14 isoparaffin/ Laureth-7(Sepigel 305)  0.2 g Sodium EDTA 0.01 g Chlorobutanol  0.4 g Water   6 g

Example 4b

Coated Tablets

Tablet Cores: Atorvastatin lactone 100 g Atorvastatin calcium 100 gDibasic calcium phosphate dihydrate 140 g Microcrystalline cellulose  24g Sodium starch glycolate  10 g Magnesium stearate  1 g Water  6 gCoating: Azopolymer solution 10% w/v in water

Example 4c

Coated Tablets of Enteric-coated Granules

Tablet Cores: Atorvastatin lactone 100 g Pitavastatin calcium 100 gCellulose acetate phthalate solution 10% w/v in acetone Acacia solution10% w/v in water Coating: Galactomannan solution 10% w/v in water

Example 4d

Hydrophilic Ointment USP.

Atorvastatin lactone 1.0 g Naproxen sodium 1.0 g Methylparaben 0.025 gPropylparaben 0.015 g Sodium lauryl sulfate 1.0 g Propylene glycol 12 gStearyl alcohol 25 g White petrolatum 25 g Purified water 35 g

Example 4e

Polyethylene Glycol Ointment NF.

Atorvastatin lactone 1.0 g Diclofenac 1.0 g Polyethylene glycol 3350  40g Polyethylene glycol 400  58 g

Example 4f

Rectal Suppository.

Atorvastatin lactone 0.10 g Aminosalicylic acid 0.90 g Theobroma oil 1.0 cc

Example 4g

Ointment in Hydrophilic Petrolatum USP.

Atorvastatin lactone 1.5 g Indomethacin 1.5 g Cholesterol 3.0 g Stearylalcohol 3.0 g White wax 8.0 g White petrolatum  86 g

Example 4h

Gel Formulation

Atorvastatin lactone 0.1 g Indomethacin 0.1 g Polyglyceryl acrylate(Norgel) 3 g Polyacrylamide/C13–14 isoparaffin/ 0.2 g Laureth-7 (Sepigel305) Sodium EDTA 0.01 g Chlorobutanol 0.4 g Water 6 g

Example 4i

Cream Formulation

Phase A: Atorvastatin lactone 1 g Indomethacin 1 g 5-n-Octanoylsalicylic acid 0.5 g Sweet almond oil 14.5 g Karate butter 7 g PPG-3myristyl ether 5 g Propyl paraben 0.1 g Polysorbate 60 2.5 g Sorbitanstearate 2.5 g Phase B: Cyclomethicone 4 g Xanthan gum 0.2 gCarboxyvinyl polymer 0.5 g Phase C: Triethanolamine 0.5 g Water 2 gPhase D: Methyl paraben 0.2 g Glycerol 5 g Water 54.5 g

Example 4j

Atorvastatin Lactone Skin Ointment in Petrolatum USP Ointment.

Atorvastatin lactone 2.0 g Petrolatum USP  98 g

Example 4k

Fluvastatin Lactone Skin Ointment in Hydrophilic Petrolatum USP.

Fluvastatin lactone 3.0 g Cholesterol 3.0 g Stearyl alcohol 3.0 g Whitewax 8.0 g White petrolatum  86 g

Example 4l

Cerivastatin Lactone Skin ointment in Hydrophilic Ointment USP.

Cerivastatin lactone 0.5 g Methylparaben 0.025 g Propylparaben 0.015 gSodium lauryl sulfate 1.0 g Propylene glycol 12 g Stearyl alcohol 25 gWhite petrolatum 25 g Purified water 37 g

Example 4m

Pitavastatin Lactone Skin Ointment in Polyethylene Glycol Ointment NF.

Pitavastatin lactone 2.0 g Polyethylene glycol 3350  40 g Polyethyleneglycol 400  60 g

Example 4n

Isotonic Rosuvastatin Calcium Solution for Ocular Use.

Rosuvastatin calcium 1.0 g Sodium chloride USP 0.9 g Benzalkoniumchloride 0.01 g Sterile distilled water to 100 mL

Example 4o

Cerivastatin Lactone Ointment for Ocular Use.

Cerivastatin lactone  2.0 g White petrolatum 97.5 g Chlorobutanol 0.50 g

Example 4p

Ointment for Ocular Use.

Atorvastatin lactone  1.0 g Cromolyn sodium  1.0 g White petrolatum 97.5g Chlorobutanol 0.50 g

Example 4q

Fluvastatin Sodium Solution for Otic use.

Fluvastatin sodium 1.0 g Sodium dihydrogen phosphate 0.56 g Disodiumhydrogen phosphate 0.28 g Sodium chloride 0.5 g Disodium edetate 0.1 gPhenyl mercuric nitrate 0.005 g Glycerin 30 mL Sterile distilled waterto 100 mL

Example 4r

Cerivastatin Sodium Retention Enema.

Cerivastatin sodium 0.40 g Sodium dihydrogen phosphate 1.6 g Disodiumhydrogen phosphate 17.9 g Sodium chloride 36 g Sodium ascorbate 2.0 gTragacanth 16 g Methylparaben 8 g Propyl paraben 2 g Propylene glycol100 mL Distilled water to 4000 mL

Example 4s

Pitavastatin Lactone Retention Enema.

Pitavastatin lactone 0.010 g Sodium carboxymethyl 1.0 g cellulose USPDistilled water 100 mL

Example 4t

Fluvastatin Lactone Rectal Suppository.

Fluvastatin lactone 0.10 g Theobroma oil 2.0 cc

Example 4u

Atorvastatin Lactone Rectal Suppository.

Atorvastatin lactone 0.10 g Polyethylene glycol 1000 1.5 g Polyethyleneglycol 4000 0.5 g

Example 4v

Pitavastatin Lactone Dry Powder Aerosol Formulation

Pitavastatin lactone 0.004 g Lactose 0.0085 g(The mixture is micronized to mass median particle size between 3–6 μm)

Example 4w

Fluvastatin Sodium Metered-dose Aerosol Formulation

Fluvastatin sodium 0.080 g (Micronized to mass median particle sizebetween 3–6 μm) Ethanol USP 0.20 g Dichlorodifluoromethane 19.72 g(Propellant)

Example 4x

Dry Powder Aerosol Formulation

Atorvastatin lactone 0.004 g Cromolyn sodium 0.004 g Lactose 0.0085 g(The mixture is micronized to mass median particle size between 3–6 μm)

Example 4y

Metered-dose Aerosol Formulation

Atorvastatin lactone 0.040 g Cromolyn sodium 0.04 g (Micronized to massmedian particle size between 3–6 μm) Ethanol USP 0.20 gDichlorodifluoromethane 19.72 g(Propellant)

Example 5

Human p38α MAP Kinase Inhibition Assay.

In vitro cell-free p38α MAP kinase inhibition assays were conducted bythe method as described in Clerk et al., FEBS Lett., 426:93–96 (1998)for a number of lactones in formulas I/Ia/IIb. Briefly, humanrecombinant p38α protein kinase expressed in E. coli (UBI #14-251) wasused. Myelin basic protein (MBP, UBI #13-110) was employed as substrate,and microtiter plate wells were coated with MBP (0.01 mg/ml) overnightat 4° C. Candidate compound and/or vehicle was preincubated with 0.075μg/mL enzyme in modified HEPES buffer pH 7.4 at 25° C. for 15 minutes.The reaction was initiated by addition of 100 μM ATP and allowed toproceed for another 60 minutes. The reaction was terminated byaspirating the solution. Phosphorylated MBP was detected by incubationwith a mouse monoclonal IgG2a anti-phosphoMBP antibody. Boundanti-phosphoMBP antibody was quantitated by incubation with a HRPconjugated goat anti-mouse IgG. The protein kinase activity wasproportional to the readings of optical density at 405 nm resulting fromreaction with an ABTS Microwell Peroxidase Substrate System.

Using this method, IC₅₀ data was obtained, with results illustrated inTable I, as discussed above.

Example 6

Whole Cell Anti-inflammation Assay.

The procedure as described in Welker et al., Int. Arch. Allergy &Immunology 109:110–115 (1996) can be followed. That is, peripheral bloodmononuclear cells (PBMCs) can be prepared from four different donors bydifferential centrifugation on Ficoll-Hypaque (Seromed, Berlin,Germany). Two donors (1 and 2) may have seasonal rhino-conjunctivitis,e.g., with positive prick tests to inhalant allergens and elevated serumIgE levels. PBMCs may contain approximately 10% CD14-positive monocyticcells, approximately 90% lymphocytes and approximately <1% granulocytesand platelets.

THP-1 cells are obtained from the ATCC (Rockville, Md., USA; TIB 202)and can be routinely kept in RPMI medium (Gibco, Eggenstein, Germany)with 10% FCS (Seromed) and 50 μM mercaptoethanol (Gibco) added. HL-60cells (ATCC; No. CCL 240) can be kept in RPMI medium, with 20% FCS, andU-937 cells (ATCC; No. CCL 1593) can be kept in RPMI medium with 10%FCS.

The following glucocorticoids are dissolved in DMSO: Methylprednisoloneaceponate (MPA), methylprednisolone-17-propionate (MPP), prednicarbate(PC) and betamethasone valerate (BMV) (Schering, Berlin, Germany). Thestock solutions are diluted with medium to <0.1% DMSO before use toavoid toxic effects on the cells.

All cells (10⁶/ml) can be kept in 24-well polystyrene culture plates andstimulated with lipopolysacharide (LPS; 50 ng/ml; Sigma, St. Louis, Mo.,USA) for 24 h at 37° C. in RPMI medium (Gibco) without serum, alone orwith 10⁻⁵-⁻⁸ M GC added.

THP-1, HL-60 and U-937 cells can also be stimulated with a combinationof phorbol myristate acetate (PMA; 25 ng/ml) and the calcium ionophoreA23187 (Ion; 2×⁻⁷ M; both from Sigma). In pre-incubation experiments,cells are cultured for 1 h with the different GCs (10⁻⁶ M) beforeaddition of the stimulus. As controls, cells are cultured with mediumonly, without stimulus or GCs and with 0.1% DMSO. After incubation,cells are centrifuged, and the culture supernatants frozen at −20° C.until analysis.

Cytokines (IL-1β, 1L-8 and TNF-α) in cell supernatants can be quantifiedby ELISA (Quantikine, Biermann, Bad Nauheim, Germany), and data can beexpressed as means of two values calculated for 10⁶ viable cells. Dataof duplicate measurements may fluctuate within a very narrow margin(<5%). All experiments can be repeated three (cell lines) or four (PBMC)times. 5×10⁷ THP-1 cells stimulated for 24 h with or without PMA/A23187and with or without 10⁻⁶ M MPA can be lysed with 3 M lithium chlorideand 6 M urea, centrifuged at 20,000 rpm for 60 min, and RNA extracted inphenol-chloroform.

8 μg total RNA per lane can be electrophorased and transferred tonitrocellulose membranes (NEN Research, Boston, Mass., USA) by standardtechniques. For Northern blot hybridization, HinIII/Bam-HI DNA fragmentsof TNF-α (680 bp) can be used. The fragments can be nick translatedusing ³²P-labeled dCTP (NEN Research) and a random primer labeling kit(Boehringer, Mannheim, Germany). Hybridization can be carried out in SSC(NaCl/sodium citrate) (Sigma) buffer containing 50% formamide (Sigma)and 10% dextran sulfate (Sigma) over-night at 42° C., according tostandard procedures. On the following day, nitrocellulose membranes canbe washed twice in 2×SSC buffer containing 0.1% sodium dodecyl sulfate(SDS; Sigma) for 15 min at 42° C. and twice in 0.2×SSC containing 0.1SDS at 50° C. After drying, the blot can be exposed to an X-ray film(Kodak, Rochester, Mass., USA) for up to 7 days.

Statistical significance may be calculated with the two-tailed t-test.The IC₅₀ data (inhibitory constants) may be calculated as the GCconcentration that cause 50% inhibition of cytokine release, using acomputer-assisted program (SPSS, Microsoft).

Example 7

Whole Cell LPS-Stimulated TNF-α Release Assay

The procedure as described in Welker et al., Int. Arch. Allergy andImmunol. 109:110–115 (1996) can be followed. Briefly, a candidatecompound and/or vehicle can be preincubated with human peripheral bloodmononuclear leukocytes (PBML, 5×10⁵/ml) cells in AIM-V medium pH 7.4 for2 hours. Lipopolysaccharide (LPS, 25 ng/ml) can be added to stimulatethe cells, which can be incubated overnight at 37° C. TNF-α cytokinelevels in the conditioned medium can then be quantitated using asandwich ELISA kit.

Example 8

Model for Inhibition of Topical Inflammation.

Groups of 5 BALB/c male mice weighing 22±2 g were sensitized byapplication of oxazolone (100 μL, 1.5% v/v in acetone) to the shavedabdominal surface. Seven days after sensitization, a candidate compound(0.1–5 mg in 20 μL acetone, methanol or ethanol vehicle) or vehiclealone (20 μL) was applied topically to the anterior and posteriorsurfaces of the right ear 30 minutes before and 15 minutes afteroxazolone (1% v/v, 25 μL/ear) challenge was applied in the same mannerto the right ear. Left ears were untreated. The thickness of both earsof each animal was measured with a Dyer model micrometer gauge 24 hoursafter oxazolone challenge, and the net increase in thickness of rightears versus left ears was calculated for each animal. Percent inhibitionwas calculated according to the formula: [(Iv−It)/Iv]×100, where Iv andIt respectively refer to the average net increase in right ear thickness(mm) for vehicle and candidate compound treated mice.

Table II summarizes the results obtained using statin lactones, saltforms of hydroxy acid statins, indomethacin, and combinations thereofusing this method.

TABLE II Results of inhibition of oxazolone-induced mouse ear swellingby statin lactones, indomethacin, salt forms of hydroxy acid statins,and combinations thereof. Compound Dose % Inh. Compound Dose % Inh.Atorvastatin 2 × 2 mg 50 Atorvastatin sodium 2 + 2 mg 63 lactoneAtorvastatin 2 × 1 mg 58 Atorvastatin sodium 2 × 1 mg 31 lactoneAtorvastatin 2 × 0.3 mg 40 Atorvastatin sodium 2 × 0.3 mg 9 Simvastatin2 × 0.3 mg 7 Simvastatin sodium 2 × 0.3 mg 31 lactone Rosuvastatin 2 ×0.3 mg 4 Rosuvastatin sodium 2 × 0.3 mg 20 lactone Pitavastatin 2 × 2 mg4 Pitavastatin calcium 2 × 2 mg 60 lactone Pitavastatin 2 × 0.3 mg 24Pitavastatin calcium 2 × 0.3 mg 9 lactone Fluvastatin lactone 2 × 0.3 mg15 Fluvastatin sodium 2 × 0.3 mg 28 Pravastatin sodium 2 × 0.3 mg 24Indomethacin 2 × 0.3 mg 44 Atorvastatin lactone + 2 × 0.3 mg 64Indomethacin

Table II shows a 50%, 58% or 40% inhibition of inflammatory ear swellingupon admininstration of atorvastatin lactone at a dose of 2×0.3 mg,while the administration of the non-statin anti-inflammatory agentindomethacin showed a 44% inhibition. Table II also shows 64% inhibitionof inflammation where the treatment comprised administration ofatorvastatin lactone and indomethacin. The 64% inhibition is an exampleof a synergistic effect by the combination of the statin lactone and thenon-statin anti-inflammatory agent.

Several observations may be made from this data. First, atorvastatinlactone exhibits the most potent anti-inflammatory activity in thisassay, with 40% inhibition of ear swelling at a dose of 2×0.3 mg.Second, atorvastatin lactone displays similar potency to thenon-steroidal anti-inflammatory drug indomethacin. Third, a combinationof atorvastatin lactone and indomethacin is more effective at reducingear swelling than either agent alone. Fourth, atorvastatin sodium andpitavastatin calcium effectively inhibit ear swelling by 63% and 60%,respectively, at a dose of 2×2 mg.

Example 9

Scheme for Side Chain Synthesis

An overall scheme for synthesizing the lactone/hydroxy acid hydroxy sidechains of compounds of formula I/II is provided below. Two approachesfor carrying out the overall scheme are detailed in Examples 9a and 9b.

Example 9a

Approach 1 for Carrying Out Side Chain Synthesis Scheme

In one approach, the structure indicated below is used in attaching theside chain and is prepared in eight steps (steps 1–8)

Step 1: ethyl 5-hydroxy-3-oxo-7-(trimethylsilyl)hept-6-ynoate, 1, isprepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under constant nitrogen flow. To a solution ofdiisopropylamine (174.3 mL) in tetrahydrofuran (1500 mL) at −78° C. isadded n-butyllithium (2.5M in hexanes, 666.6 mL) via cannula. Themixture is warmed to −40° C. for 0.5 h (h), then cooled back to −78° C.and ethyl acetoacetate (104.3 g) is added neat, dropwise. The coolingbath is removed and the mixture left to warm for 1 h, then re-cooled to−78° C. and the aldehyde (103.0 g) is added neat, dropwise. Afterallowing the reaction to warm to ambient temperature over 16 h, themixture is partitioned between ethyl acetate and water, acidifying theaqueous layer to pH 2. The aqueous layer is extracted with furtherportions of ethyl acetate and the combined organics washed with brinetwice, dried over magnesium sulfate, filtered, and concentrated to abrown oil (266 g) which is used crude in the next step.

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 0.17(s, 9H), 1.26(t, 3H), 2.97–3.00(m, 2H); 3.50(s,2H), 4.18–4.23(q, 2H), 4.82(dd, 1H).

Step 2: ethyl 3,5-dihydroxy-7-(trimethylsilyl)hept-6-ynoate, 2, isprepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under constant nitrogen flow. A solution of crudeethyl 5-hydroxy-3-oxo-7-(trimethylsilyl)hept-6-ynoate, 1 in a mixture ofanhydrous tetrahydrofuran (2000 mL) and anhydrous methanol (220 mL) iscooled to −78° C. and diethylmethoxyborane (107.3 mL) is added neat,dropwise. The mixture is left to stir for 1 h then sodium borohydride(30.9 g) is added portionwise. The mixture is left to warm to ambienttemperature over 16 h, then cooled in an ice-bath and acetic acid (170g) added. After stirring for 0.5 h, the reaction mixture is partitionedbetween ethyl acetate and aqueous sodium hydrogen carbonate and theorganic layer washed until pH 8, washed with brine, dried over magnesiumsulfate, filtered and concentrated to a brown oil. This oil is dissolvedin methanol, and concentrated in vacuo at 60° C. After 3 furtherrepetitions of this, the crude title compound (152 g) is taken on to thenext step.

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 0.17(s, 9H), 1.26(t, 3H), 1.82–1.96(m, 2H),3.06(brs, 1H), 3.58(brs, 1H), 4.17(q, 2H), 4.21–4.29(m, 1H),4.60–4.67(dd, 1H).

Step 3: ethyl2-(2,2-dimethyl-6-((trimethylsilyl)ethynyl)-1,3-dioxan-4-yl)acetate, 3,is prepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under constant nitrogen flow. A solutioncontaining crude ethyl 3,5-dihydroxy-7-(trimethylsilyl)hept-6-ynoate, 2(152.0 g), 2,2-dimethoxypropane (362.0 mL), p-toluenesulphonic acidmonohydrate (56.0 g) and 3 Å molecular sieves (200 g) in anhydrousethyleneglycol dimethylether (1100 mL) is shaken at ambient temperaturefor 16 h. The reaction mixture is diluted with dichloromethane andpartitioned versus aqueous sodium hydrogen carbonate. The organic layeris washed with brine, dried over magnesium sulfate, filtered andconcentrated. Purification by flash chromatography affords the titlecompound (89.6 g) as an oil.

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 0.17(s, 9H), 1.26(t, 3H), 1.43(s, 3H), 1.48(s, 3H),1.52°1.89(m, 2H), 2.35–2.62(m, 2H), 4.16(q, 2H), 4.22–4.37(m, 1H),4.70(dd, 1H).

Step 4: 2-(6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetic acid, 4, isprepared as follows:

To a solution of ethyl2-(2,2-dimethyl-6-((trimethylsilyl)ethynyl)-1,3-dioxan-4-yl)acetate, 3(30.0 g) in ethanol (155 mL) at 0° C. is added a solution of sodiumhydroxide (2 M, 105.7 mL) dropwise. After 3 h of stirring at ambienttemperature, the mixture is partitioned between ethyl acetate and water.The aqueous layer is washed with 3 portions of ethyl acetate and theorganics discarded. The aqueous layer is acidified to pH 4 and extractedwith 4 portions of ethyl acetate. The combined organics are dried overmagnesium sulfate, filtered and concentrated to afford the titlecompound (16.6 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.43(s, 3H), 1.47(s, 3H), 1.61–1.95(m, 2H),2.45–2.65(m, 3H), 4.23–4.40(m, 1H), 4.68–4.72(m, 1H).

Step 5: (R)-1-(naphthalen-1-yl)ethanaminium, (4R,6S)- and(4S,6R)-2-(6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate salt, 5, isprepared as follows:

To a cooled solution of 2-(6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)aceticacid, 4 (63.4 g) in diethyl ether (200 mL) at 0° C. is added a solutionof (R)-1-(naphthalen-1-yl)ethanamine (54.9 g) in diethyl ether (200 mL).The mixture is stirred for 0.5 h and the solvent is removed in vacuo togivea mixture of the diastereomeric salts, 5 (117.0 g).

Step 6: (R)-1-(naphthalen-1-yl)ethanaminium,2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate salt, 6, isprepared as follows:

The crude salt, 5 (117 g) is recrystallized from hot methyl isobutylketone at a concentration of 45 mg/mL. The resulting crystals arefiltered, washing with ice-cold methyl isobutyl ketone then hexanes anddried in vacuo. This process is repeated twice more, at which point thematerial is diastereomerically pure as assessed by chiral HPLC(Chiralpak AD column, eluting with 1–8% ethanol in hexane) and by ¹H NMR(29.8 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.30(s, 3H), 1.32(s, 3H), 1.63 (d, 3H), 1.24–1.71(m,2H), 2.07(ddd, 2H), 2.45(s, 1H), 3.95–4.05(m, 1H), 4.48(m, 1H),5.03–5.13(q, 1H), 6.20–6.65(brs, 3H), 7.48–7.62(m, 3H), 7.66(d, 1H),7.79(d, 1H), 7.88(d, 1H), 8.03(d, 1H).

Step 7: 2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetic acid,7, is prepared as follows:

(R)-1-(naphthalen-1-yl)ethanaminium,2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate salt, 6 (6.58g) is partitioned between ethyl acetate and dilute hydrochloric acid(0.2 M, 89.2 mL). The organic layer is dried over magnesium sulfate,filtered and concentrated to give2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetic acid, 7 as ayellow oil (3.60 g) which is used without further purification.

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.43(s, 3H), 1.47(s, 3H), 1.61–1.95(m, 2H),2.45–2.65(m, 3H), 4.23–4.40(m, 1H), 4.68–4.72(m, 1H).

Step 8: ethyl 2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate,8, is prepared as follows:

To a solution of2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetic acid, 7 (3.60g) in anhydrous acetonitrile (40 mL) is added a solution of iodoethane(4.25 g) in anhydrous acetonitrile (10 mL) followed by a solution of1,8-diazabicyclo[5.4.0]undec-7-ene (4.15 g) in anhydrous acetonitrile(10 mL). The mixture is warmed to 55° C. for 1.5 h, then cooled and thesolvent is removed in vacuo. The residue is partitioned between ethylacetate and aqueous sodium hydrogen carbonate. The organic layer iswashed with 0.05 M hydrochloric acid solution, then brine and dried overmagnesium sulfate, filtered and concentrated to an oil (3.01 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.26(t, 3H), 1.43(s, 3H), 1.47(s, 3H), 1.50–1.95(m,2H), 2.34–2.64(m, 3H), 4.16(q, 2H), 4.20–4.40(m, 1H), 4.66–4.72(m, 1H).

Example 9b

Approach 2 for Carrying Out Side Chain Synthesis Scheme

In a second approach, the structure indicated below is used in attachingthe side chain. This structure can be prepared in one step (Step 1) fromthe product obtained in Example 9a, as detailed below.

Step 1: ethyl 2-((4S,6R)-2,2-dimethyl-6-vinyl-1,3-dioxan-4-yl)acetate,9, is prepared as follows:

To a solution of ethyl2-((4R,6S)-6-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate, 8 (0.70 g) inethanol (15 mL) is added ammonium formate (1.95 g), quinoline (13 μL)and palladium on calcium carbonate (0.07 g). The reaction mixture isheated to reflux with periodical additions of ammonium formate andpalladium on calcium carbonate until the reaction is complete. Thereaction is then filtered through Celite and partitioned between ethylacetate and water. The organic phase is separated, dried over magnesiumsulfate, filtered again and evaporated to afford the title compound(0.62 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.26(t, 3H), 1.43(s, 3H), 1.48(s, 3H), 1.30–1.70(m,2H), 2.46(ddd, 2H), 4.12–4.19(m, 2H), 4.33–4.41(m, 2H), 5.12(d, 1H),5.28(d, 1H), 5.77–5.86(m, 1H).

Example 10

Scheme for Synthesis of N-pyridyl Pyrazole Compounds

An overall scheme for synthesizing N-pyridyl substituted pyrazolecompounds of formula VI of the instant invention is provided below.Examples 10a, 10b, 10c and 10d below detail four specific examplesfollowing the overall scheme.

Example 10a

Synthesis of an N-pyridyl Pyrazole

In one specific example, a compound having the structure indicated belowis prepared in nine steps (Steps 1–9).

Step 1: 1-(4-fluorophenyl)-4-methylhexane-1,3-dione, 10, is prepared asfollows:

To a 0° C. suspension of sodium hydride (15 g) in anhydrous 1,4-dioxane(150 mL) under nitrogen is added dropwise a solution of1-(4-fluorophenyl)ethanone in 1,4-dioxane (50 mL), followed by dropwiseaddition of a solution of ethyl-2-methylbutanoate in 1,4-dioxane (50mL). The resulting solution is heated to 80° C. for 4 h during whichtime vigorous gas evolution occurs. The reaction mixture is poured intoiced 1N hydrochloric acid, then extracted twice with ethyl acetate. Thecombined organic layers are washed with brine, then dried over magnesiumsulfate, filtered and concentrated. Distillation under reduced pressureaffords 12.4 g of the title compound as an oil.

The compound obtained in this step shows the following boiling point andmass spectral data:

b.p. 101–104° C. at 1 mm Hg

LC/MS: C₁₃H₁₅FO₂ requires 222.1; observed M/Z 221.3 [M−H]⁻. RT (RT) 5.62min (min).

Step 2: 4-hydrazinylpyridin-2(1H)-one, 11, is prepared as follows:

To a suspension of 4-hydroxypyridin-2(1H)-one (5 g) in 2-ethoxyethanol(20 mL) is added hydrazine (10 mL). The mixture is flushed withnitrogen, then heated to reflux for 4 days. Upon cooling to 0° C., thetitle compounds forms a precipitate which is filtered, then taken up inboiling ethanol and re-filtered, washing with further hot ethanol.Removal of the solvent in vacuo yields 2.2 g of the title compound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₅H₇N₃O requires 125.1; observed M/Z 126.1 [M+H]⁺. RT 0.50min.

Step 3:4-(3-sec-butyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)pyridine-2(1H)-one,12a and4-(5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)pyridine-2(1H)-one,12b are prepared as follows:

A solution of 1-(4-fluorophenyl)-4-methylhexane-1,3-dione, 10 (0.200 g)and 4-hydrazinylpyridin-2(1H)-one, 11 (0.113 g) is dissolved in aceticacid (3 mL) and the mixture heated to 90° C. for 2 h. On cooling, thesolution is quenched with sodium hydrogen carbonate and extracted withethyl acetate. The aqueous layer is extracted with a further portion ofethyl acetate and the combined organic layer is dried over magnesiumsulfate, filtered and concentrated to an oil that crystallizes onstanding. The residue is triturated with 9:1 iso-hexanes/ethyl acetateto give 0.145 g of 12a and 12b as a mixture.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₈FN₃O requires 311.1; observed M/Z 312.2 [M+H]⁺, 310.3[M−H]⁻. RT 5.25 min.

Step 4:4-(5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13aand 4-(3-sec-butyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine,13b are prepared as follows:

The mixture of regioisomers, 12a and 12b (5.3 g) are dissolved inphosphorous oxychloride (30 mL) and heated to 95° C. for 16 h. Oncooling the reaction mixture is poured into ice-water, then extractedwith ethyl acetate. The organic layer is washed with aqueous sodiumhydrogen carbonate and brine then dried over magnesium sulfate, filteredand concentrated. Flash chromatography affords 2.77 g of4-(5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13aand 0.54 g of4-(3-sec-butyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13bas well as 0.57 g of mixed fractions.

4-(5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13ashows the following mass spectral and NMR data:

LC/MS: C₁₈H₁₇ClFN₃ requires 329.1; observed M/Z 330.1/332.0 (Cl) [M+H]⁺.RT 7.40 min

¹H-NMR (270 MHz, CDCl₃, δ) 0.96(t, 3H), 1.30(d, 3H), 1.52–1.80(m, 2H),277–2.89(m, 1H), 6.31(s, 1H), 7.03(d, 1H), 7.09–7.15(m, 2H),7.21–7.28(m, 2H), 7.41(s, 1H), 8.22(d, 1H).

4-(3-sec-butyl-5-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13bshows the following mass spectral data:

LC/MS: C₁₈H₁₇ClFN₃ requires 329.1; observed M/Z 330.1/332.0 (Cl) [M+H]⁺.RT 7.40 min

Step 5:4-(4-bromo-5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine,14 is prepared as follows:

A solution of4-(5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine, 13a(2.49 g) and N-bromosuccinimide (2.69 g) are dissolved inN,N-dimethylformamide (40 mL) and stirred for 3 h at room temperature.The crude mixture is partitioned between water and dichloromethane andthe aqueous phase is extracted with further portions of dichloromethane.The combined organic layers are washed with brine, dried over magnesiumsulfate, filtered and concentrated. Flash chromatography affords 2.54 gof the title compound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₆BrClFN₃ requires 407.0; observed M/Z407.8/409.9/411.9 (Br/Cl) [M+H]⁺. RT 8.21 min

Step 6:4-(4-bromo-5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-phenylpyridin-2-amine,15 is prepared as follows:

To a solution of4-(4-bromo-5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-chloropyridine,14 (2.53 g) and aniline (1.13 g) in 2,2,2-trifluoroethanol (20 mL) isadded trifluoroacetic acid (2.21 mL) dropwise and the mixture heated to80° C. for 36 h. On cooling the reaction mixture is partitioned betweensodium hydrogen carbonate and ethyl acetate. The organic layer is washedwith brine, dried over magnesium sulfate, filtered and concentrated.Purification by flash chromatography provides 1.10 g of the titlecompound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₄H₂₂BrFN₄ requires 464.1; observed M/Z 464.9/466.9 (Br)[M+H]⁺, 463.0/465.0 (Br) [M−H]⁻. RT 7.72 min

Step 7: Ethyl2-((4R,6S)-6-((E)-2-(5-sec-butyl-3-(4-fluorophenyl)-1-(2-(phenylamino)pyridine-4-yl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,16 is prepared as follows:

To a 0° C. solution of ethyl2-((4R,6S)-ethynyl-2,2-dimethyl-1,3-dioxan-4-yl)acetate, 9 (1.34 g) anddichlorobis(triphenylphosphine)palladium(II) (0.040 g) under nitrogen inanhydrous tetrahydrofuran (3 mL) is added neat tributyltin hydride (1.79g) dropwise. The resulting mixture is stirred for 30 min, then added toa solution containing4-(4-bromo-5-sec-butyl-3-(4-fluorophenyl)-1H-pyrazol-1-yl)-N-phenylpyridin-2-amine,15 (1.10 g) and dichlorobis(triphenylphosphine)palladium(II) (80 mg) inN,N-dimethylformamide (4 mL). The reaction mixture is flushed withnitrogen and heated to 80° C. for 16 h. On cooling, the mixture ispartitioned between ethyl acetate and brine and the organic layer washedwith further brine, dried over magnesium sulfate, filtered andconcentrated. Purification by flash chromatography affords 0.57 g of thetitle compound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₆H₄₁FN₄O₄ requires 612.3; observed M/Z 613.1 [M+H]⁺,611.3 [M−H]⁻. RT 7.60 min.

Step 8: (3R,5S,E)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,17 is prepared as follows:

To a 0° C. solution of ethyl2-((4R,6S)-6-((E)-2-(5-sec-butyl-3-(4-fluorophenyl)-(2-(phenylamino)pyridine-4-yl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,16 (0.640 g) in 3:1 tetrahydrofuran/water (10 mL) is addedp-toluenesulfonic acid monohydrate (0.298 g) and the mixture stirred atroom temperature for 72 h. The reaction mixture is partitioned betweenethyl acetate and brine, the organic layer is washed with sodiumhydrogen carbonate and brine, dried over magnesium sulfate, filtered andconcentrated. Purification by flash chromatography affords 0.270 g ofthe title compound.

The compound obtained in this step shows the following mass spectraldata:

LC/MS: C₃₃H₃₇FN₄O₄ requires 572.3; observed M/Z 573.2 [M+H]⁺, 571.2[M−H]⁻. RT 5.79 min.

Step 9:(3R,5S,E)-7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoicacid calcium salt, 18 is prepared as follow:

To a 0° C. solution of (3R,5S,E)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,17 (0.135 g) in ethanol (0.6 mL) is added a 1M solution of sodiumhydroxide (0.236 mL) dropwise and the mixture stirred at roomtemperature for 2 h. Ethanol is removed in vacuo until the compoundstarts to precipitate, then an aqueous solution of calcium chloride(0.118M, 2.0 mL) is added dropwise. The resulting precipitate isfiltered, washed with water, acetonitrile, water, acetonitrile and driedin vacuo to afford the title compound (0.077 g).

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS as free acid: C₃₁H₃₃FN₄O₄ requires 544.2; observed M/Z 545.1[M+H]⁺, 545.0 [M−H]⁻. RT 2.99 min.

¹H-NMR (270 MHz, DMSO-d₆, δ) 0.91(t, 3H), 1.28(d, 3H), 1.20–1.32(m,2H)1.48–1.59(m, 2H), 1.80–2.12(m, 2H), 2.93–3.01(m, 1H), 3.69–3.76(m,1H), 4.09–4.15(m, 1H), 4.92(brs, 1H), 5.64(dd, 1H), 6.19(d, 1H), 6.30(d,1H), 6.80(s, 1H), 6.89(t, 1H), 7.21(t, 2H), 7.29–7.36(m, 4H), 7.48(2H),8.02(d, 1H), 9.15(s, 1H).

Example 10b

Synthesis of an N-pyridyl Pyrazole

In a second specific example, a compound having the structure indicatedbelow is prepared in one step (Steps 1) from the product obtained inExample 10a, detailed above.

Step 1:(4R,6R,E)-6-(2-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridine-4-yl)-1H-pyrazol-4-yl)vinyl)-4-hydroxy-tetrahydropyran-2-one,19 is prepared as follows:

(3R,5S,E)-7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoicacid calcium salt, 18 (0.050 g) is dissolved in toluene (2.5 mL) andheated to 90° C. for 10 h. The solution is concentrated in vacuo andpurified by flash chromatography to give 0.026 g of the title compoundas a powder.

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS: C₃₁H₃₁FN₄O₃ requires 526.2; observed M/Z 527.1 [M+H]⁺, 525.2[M−H]⁻. RT 5.59 min.

¹H-NMR (270 MHz, CDCl₃, δ) 0.97(t, 3H), 1.33(d, 3H), 1.48–2.05(m, 4H), 22.80(m, 2H), 2.87–2.96(m, 1H), 4.3–4.37(m, 1H), 5.11–5.14(m, 1H),5.62(dd, 1H), 6.31(d, 1H), 6.48–6.54(m, 2H), 6.77(d, 1H), 6.88(d, 2H),7.05(t, 1H), 7.09–7.32(m, 5H), 8.08(d, 1H).

Examples 10c and 10d

Syntheses of N-pyridyl Pyrazoles

In two additional specific examples, compounds having the structuresindicated below are prepared in two steps (Steps 1–2) from intermediate17 obtained in Example 10a above.

Step 1: (3R,5R)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoate,20 and(4R,6R)-6-(2-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,21 is prepared as follows:

A flask containing (3R,5S,E)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,17 (0.100 g), ammonium formate (0.220 g) and palladium on carbon (0.020g) in ethanol (5 mL) is heated to 80° C. for 3 h, adding furtherportions of ammonium formate and catalyst every 1 h. The resultingmixture is filtered through celite and concentrated in vacuo, thenpurified by flash chromatography to furnish 0.043 g of (3R,5R)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoate,20 and(4R,6R)-6-(2-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,21 (0.002 g).

(3R,5R)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydrokyheptanoate,20 shows the following mass spectral data: C/MS: C₃₃H₃₉FN₄O₄ requires574.3; observed M/Z 575.1 [M+H]⁺. RT 5.85 min.

(4R,6R)-6-(2-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,21 shows the following mass spectral data: LC/MS: C₃₁H₃₃FN₄O₃ requires528.3; observed M/Z 529.1 [M+H]⁺. RT 5.50 min.

Step 2:(3R,5R)₇7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoicacid calcium salt, 22 is prepared as follows:

(3R,5R)-7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoicacid calcium salt, 22 (0.026 g) is obtained as a powder in the samemanner as Example 10a, Step 9 from (3R,5R)-ethyl7-(3-sec-butyl-5-(4-fluorophenyl)-1-(2-(phenylamino)pyridin-4-yl)-1H-pyrazol-4-yl)-3,5dihydroxyheptanoate, 20 (0.043 g).

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS: C₃₁H₃₅FN₄O₄ requires 546.3; observed M/Z 547.2 [M+H]⁺. RT 3.07min.

¹H-NMR (270 MHz, DMSO-d₆, δ) 0.90(t, 3H), 1.25(d, 3H), 1.23–1.51(m, 4H),1.52–1.64(m, 1H), 1.70–1.82(m, 1H), 1.82–2.12(m, 2H), 2.34(m, 1H),2.72–2.85(m, 1H), 3.45–3.55(m, 1H), 3.70–3.85(m, 1H), 4.55–4.69(m, 1H),6.26(d, 1H), 6.83(s, 1H), 6.88(t, 1H), 7.15(t, 2H), 7.25–7.36(m, 4H),7.53(d, 2H), 7.95(d, 1H), 9.11(s, 1H).

Example 11

Scheme for Synthesis of N-pyrimidinyl Pyrazole Compounds

An overall scheme for synthesizing N-pyrimidinyl substituted pyrazole:compounds of formula VI of the invention is provided below. Examples11a–g and 11i below detail eight specific examples following the overallscheme.

Example 11a

Synthesis of an N-pyrimidinyl Pyrazole

In one specific example, a compound having the structure indicated belowis prepared in 8 steps (Steps 1–8).

Step 1: 3,5-diphenyl-1H-pyrazole, 22 is prepared as follows:

Hydrazine monohydrate (2.16 mL) is added to a solution of1,3-diphenylpropane-1,3-dione (10.0 g) in ethanol (100 mL) and themixture is heated to 60° C. for 2.5 h forming a white precipitate overthis time. Ethanol is removed in vacuo and the residue is taken up inethyl acetate. The resulting suspension is filtered to give3,5-diphenyl-1H-pyrazole, 22 (3.44 g) as a white powder. The filtrate iswashed with water (2×100 mL), dried over magnesium sulfate andconcentrated to give further 3,5-diphenyl-1H-pyrazole, 22 (5.90 g) as asolid.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₅H₁₂N₂ requires 220.1; observed M/Z 221.3 [M−H]⁻, 219.4[M−H]⁻. RT 4.84 min.

Step 2: 4-(3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylthio)pyrimidine, 23 isprepared as follows:

A solution of 3,5-diphenyl-1H-pyrazole, 22 (9.34 g) in anhydrousN,N-dimethylformamide (71 mL) is added dropwise to a suspension ofsodium hydride (1.87 g of a 60% dispersion in mineral oil) inN,N-dimethylformamide (36 mL) under nitrogen. After addition iscomplete, a solution of 4-chloro-2-(methylthio)pyrimidine (6.82 g) inanhydrous N,N-dimethylformamide (24 mL) is added dropwise and thereaction mixture stirred at ambient temperature for 1 h. Water is thenadded cautiously and the product extracted with ethyl acetate. Theorganic layer is washed with water, brine and dried over magnesiumsulfate, filtered and concentrated. Purification by flash chromatographyyields 8.06 g of the title compound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₆N₄S requires 344.1; observed M/Z 345.1 [M+H]⁺. RT6.60 min.

Step 3: 4-(3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,24 is prepared as follows:

To a solution of4-(3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylthio)pyrimidine, 23 (8.06 g),in a mixture of tetrahydrofuran (145 mL) and methanol (320 mL) is addeda slurry of Oxone (57.60 g) and sodium acetate trihydrate (44.64 g) inwater (80 mL). The flask is flushed with nitrogen then left to stir atambient temperature for 20 h, after which time the mixture ispartitioned between water and dichloromethane. The organic layer iswashed with brine, dried over magnesium sulfate and concentrated toafford a solid. Purification by trituration with diethyl ether furnishesthe title compound (7.70 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₆N₄O₂S requires 376.1; observed M/Z 377.1 [M+H]⁺. RT6.10 min.

Step 4:4-(4-bromo-3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,25 is prepared as follows:

To a solution of4-(3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine, 24 (3.50g) in N,N-dimethylformamide (35 mL) is added solid N-bromosuccinimide(2.49 g) and the mixture left to stir for 16 h at ambient temperature.The reaction mixture is partitioned between water and ethyl acetate,washing the organic layer with brine. The combined organic layers aredried over magnesium sulfate, filtration and concentration in vacuo, theresulting solid is triturated with diethyl ether to furnish the titlecompound (4.06 g).

The compound to be prepared at this step shows the following massspectral data: LC/MS: C₂₀H₁₅BrN₄O₂S requires 454.0; observed M/Z454.9/456.9 (Br) [M+H]⁺. RT 6.04 min.

Step 5:4-(4-bromo-3,5-diphenyl-1H-pyrazol-1-yl)-N-phenylpyrimidin-2-amine, 26is prepared as follows:

A solution of4-(4-bromo-3,5-diphenyl-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,25 (4.00 g) and aniline (1.60 mL) in dimethyl sulfoxide (40 mL) areheated to 110° C. for 60 h. The reaction mixture is then partitionedbetween ethyl acetate and brine, extracting the aqueous layer withfurther ethyl acetate. The combined organic layers are dried overmagnesium sulfate, filtered and concentrated. Purification by flashchromatography affords the title compound (1.13 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₅H₁₈BrN₅ requires 467.1; observed M/Z 467.9/469.9 [M+H]⁺.RT 6.92 min.

Step 6: ethyl2-((4R,6S)-6-((E)-2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,27 is prepared as follows:

4-(4-bromo-3,5-diphenyl-1H-pyrazol-1-yl)-N-phenylpyrimidin-2-amine, 26(0.300 g), ethyl2-((4S,6R)-2,2-dimethyl-6-vinyl-1,3-dioxan-4-yl)acetate, 9 (0.249 g) anddichlorobis-(triphenylphosphine)palladium(II) (0.012 g) are dissolved ina mixture of N,N-dimethyl-formamide (1 mL) and triethylamine (1 mL)under nitrogen and the mixture heated to 110° C. for 36 h adding 2further portions of catalyst during this time. The reaction is cooled,filtered through celite and solvent removed in vacuo. The resultingresidue is partitioned between dichloromethane and water, washing theorganic layer with brine. The organic layer is dried over magnesiumsulfate, and after filtering and concentrating, the crude residue ispurified by flash chromatography to afford the title compound (0.195 g).

Alternatively, product 27 (0.585 g) is obtained as an off-white powderin the same manner as Example 10a, Step 7 from4-(4-bromo-3,5-diphenyl-1H-pyrazol-1-yl)-N-phenylpyrimidin-2-amine, 26(0.886 g)

The compond to be obtained at this step shows the following massspectral data: LC/MS: C₃₇H₃₇N₅O₄ requires 615.3; observed M/Z 616.2[M+H]⁺, 614.4 [M−H]⁻. RT 7.34 min.

Step 7: (3R,5S,E)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,28 is prepared as follows:

The product (0.347 g) is obtained in the same manner as Example 10a,Step 8 from ethyl2-((4R,6S)-6-((E)-2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,27 (0.574 g)

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₄H₃₃N₅O₄ requires 575.3; observed M/Z 576.2 [M+H]⁺. RT5.62 min.

Step 8:(3R,5S,E)-7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoicacid calcium salt, 29 is prepared as follows:

The product (0.072 g) is obtained in the same manner as Example 10a,Step 9 from (3R,5S,E)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,28 (0.160 g)

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS as free acid: C₃₂H₂₉N₅O₄ requires 547.2; observed 548.1 [M+H]⁺,546.2 [M−H]⁻. RT 2.84 min.

¹H-NMR (270 MHz, DMSO-d₆, δ) 1.11–1.25(m, 2H), 1.86–2.12(m, 2H),3.63–3.68(m, 1H), 4.04–4.10(m, 1H), 4.84(brs, 1H), 5.46(dd, 1H), 6.33(d,1H), 6.84(t, 1H), 6.89–6.94(m, 2H), 7.05(t, 2H), 7.12(d, 1H),7.30–7.40(m, 3H), 7.42–7.54(m, 5H), 7.69(d, 2H), 8.56(d, 1H), 9.66(s,1H).

Example 11b

Synthesis of an N-pyrimidinyl Pyrazole

In a second specific example, a compound having the structure indicatedbelow is prepared in one step (Step 1) from the product obtained inExample 11a, detailed above.

Step 1:(4R,6S,E)-6-(2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)vinyl)-4-hydroxy-tetrahydropyran-2-one,30 is prepared as follows:

The product (0.018 g) is obtained in the same manner as Example 10b,Step 1 from(3R,5S,E)-7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoicacid calcium salt, 29 (0.050 g).

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS: C₃₂H₂₇N₅O₃ requires 529.2; observed 530.1 [M+H]⁺, 528.2 [M−H]⁻.RT 5.20

¹H-NMR (270 MHz, CDCl₃, δ) 1.73–1.80(m, 2H), 2.47–2.64(m, 2H),4.18–4.22(m, 1H), 5.00–5.04(m, 1H), 5.45(dd, 1H), 6.39(d, 1H), 6.70(brs,1H), 6.90(t, 1H), 6.95–7.03(m, 2H), 7.09–7.12(m, 3H), 7.30–7.41(m, 8H),7.61(d, 2H), 8.34(d, 1H).

Examples 11c and 11d

Syntheses of N-pyrimidinyl Pyrazoles

In two additional specific examples, compounds having the structuresindicated below are prepared in two steps (Steps 1–2) from intermediate28 obtained in Example 11a above.

Step 1: (3R,5R)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoate,31 and(4R,6R)-6-(2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,32 is prepared as follows:

A flask containing (3R,5S,E)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyhept-6-enoate,28 (0.227 g), ammonium formate (0.500 g) and palladium on carbon (0.050g) in ethanol (10 mL) is heated to 80° C. for 2 h adding extra portionsof catalyst and ammonium formate every 45 min. The reaction mixture isleft at ambient temperature for 16 h, then filtered through celite andconcentrated. The resulting residue is partitioned between ethyl acetateand water and the organic layer dried over magnesium sulfate, filteredand concentrated. Purification by flash chromatography affords(3R,5R)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoate,31 (0.162 g) and(4R,6R)-6-(2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,32 (0.023 g).

(3R,5R)-ethyl7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoate,31 shows the following mass spectral data:

LC/MS: C₃₄H₃₅N₅O₄ requires 577.3; observed M/Z 578.1 [M+H]⁺, 576.2[M−H]⁻. RT of 5.72 min.

(4R,6R)-6-(2-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)ethyl)-4-hydroxy-tetrahydropyran-2-one,32 shows the following mass spectral and NMR data:

LC/MS: C₃₂H₂₉N₅O₃ requires 531.2; observed M/Z 532.1 [M+H]⁺, 530.2[M−H]⁻. RT 5.34 min.

¹H-NMR (270 MHz, CDCl₃, δ) 1.22–1.83(m, 4H), 2.41–2.72(m, 2H),2.72–2.91(m, 2H), 4.18–4.21(m, 1H), 4.37–4.49(m, 1H), 6.75(s, 1H),6.93(t, 1H), 7.03(d, 2H), 7.10–7.23(m, 3H), 7.33–7.52(m, 8H), 7.73(d,2H), 8.37(d, 1H).

Step 2:(3R,5R)-7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoicacid, 33 is prepared as follows:

The product (0.090 g) is obtained in the same manner as Example 10a,Step 9 from(3R,5R)-ethyl-7-(3,5-diphenyl-1-(2-(phenylamino)pyrimidin-4-yl)-1H-pyrazol-4-yl)-3,5-dihydroxyheptanoicacid, 31 (0.162 g).

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS as free acid: C₃₂H₃₁N₅O₄ requires 549.2; observed M/Z 550.1[M+H]⁺, 548.2 [M−H]⁻. RT 3.04 min.

¹H-NMR (270 MHz, DMSO-d₆, δ) 1.12–1.42(m, 4H), 1.79–2.07(m, 2H),2.43–2.61(m, 1H), 2.64–2.80(m, 1H), 3.42–3.53(m, 1H), 3.62–3.75(m, 1H),4.56–4.67(brs, 1H), 6.82(t, 1H), 6.83–6.94(d, 2H), 6.99–7.10(t, 2H),7.15(d, 1H), 7.30–7.55(m, 8H), 7.78(d, 2H), 8.55(d, 1H), 9.59(s, 1H).

Example 11e

Synthesis of an N-pyrimidinyl Pyrazole

In a fifth specific example, a compound having the structure indicatedbelow is prepared in nine steps (Steps 1–9).

Step 1: 4-methyl-1-(3-(trifluoromethyl)phenyl)pentane-1,3-dione, 34 isprepared as follows:

The product (4.91 g) is obtained as a clear oil in the same manner asExample 10a, Step 1 from 1-(3-(trifluoromethyl)phenyl)ethanone (10.00) gand ethyl isobutyrate (6.00 g).

The compound obtained in this step shows the following boiling point andmass spectral data:

b.p 67–80° C. at 1 mm Hg

LC/MS: C₁₃H₁₃F₃O₂ requires 258.1; observed M/Z 257.3 [M−H]⁻. RT 5.09min.

Step 2: 4-hydrazinyl-2-(methylthio)pyrimidine, 35 is prepared asfollows:

To a solution of 4-chloro-2-(methylthio)pyrimidine (50.0 g) in ethanol(200 mL) is added hydrazine (15.58 g) in one portion and the mixtureleft to stir for 16 h at ambient temperature, during which time aprecipitate forms. This precipitate is filtered and washed with copiousice-cold ethanol to furnish the title compound (25.0 g) as a whitepowder.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₅H₈N₄S requires 156.0, observed M/Z 157.2 [M+H]⁺, 155.3[M−H]⁻. RT 1.79 min.

Step 3:4-(3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylthio)pyrimidine,36 is prepared as follows:

4-methyl-1-(3-(trifluoromethyl)phenyl)pentane-1,3-dione, 34 (1.65 g) and4-hydrazinyl-2-(methylthio)pyrimidine, 35 (1.00 g) are dissolved inacetic acid (15 mL) and the mixture heated to 90° C. for 2 h. Oncooling, the mixture is partitioned between dichloromethane and aqueoussodium hydrogen carbonate and the aqueous layer washed with two furtherportions of dichloromethane. The combined organics are washed withbrine, dried over magnesium sulfate, filtered and concentrated.Purification by flash chromatography affords the title compound (1.29g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₇F₃N₄S requires 378.1; observed M/Z 379.0 [M+H]⁺. RT7.43 min.

Step 4:4-(3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,37 is prepared as follows:

The product (0.485 g) is obtained in the same manner as Example 11a,Step 3 from4-(3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylthio)pyrimidine,36 (0.500 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₇F₃N₄O₂S requires 410.1; observed M/Z 411.0 [M+H]⁺. RT5.68 min.

Step 5:4-(4-bromo-3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,38 is prepared as follows:

The product (0.524 g) is obtained in the same manner as Example 11a,Step 4 from4-(3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,37 (0.496 g), after flash chromatography.

The compound obtained in this step shows the following NMR data: ¹H-NMR(400 MHz, CDCl₃, δ) 1.40(d, 6H), 2.51(s, 3H), 3.13–3.22(m, 1H), 7.58 (d,1H), 7.61–7.69(m, 2H), 7.76(d, 1H), 8.17(d, 1H), 8.88(d, 1H).

Step 6:4-(4-bromo-3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-methoxypyrimidine,39 is prepared as follows:

A mixture of4-(4-bromo-3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,38 (0.745 g) and sodium methoxide (0.087 g) in methanol (3 mL) areheated to 60° C. for 2 h. On cooling, the solvent is removed byevaporation and the residue purified by flash chromatography to furnishthe title compound (0.582 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₆BrF₃N₄O requires 440.0; observed M/Z 440.9/442.9 (Br)[M+H]⁺. RT 6.92 min.

Step 7: ethyl2-((4R,6S)-6-((E)-2-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,40 is prepared as follows:

4-(4-bromo-3-isopropyl-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-methoxy-pyrimidine,39 (0.500 g), ethyl2-((4S,6R)-2,2-dimethyl-6-vinyl-1,3-dioxan-4-yl)acetate, 9 (0.440 g) anddichlorobis(triphenylphosphine)palladium(II) (0.024 g) are dissolved ina mixture of N,N-dimethylformamide (2 mL) and triethylamine (2 mL) undernitrogen and the mixture heated to 110° C. for 36 h adding two furtherportions of catalyst during this time. The reaction is cooled, filteredthrough celite and solvent removed in vacuo. The resulting residue ispartitioned between dichloromethane and water, washing the organic layerwith brine. After drying the organic layer over magnesium sulfate,filtering and concentrating, the crude residue is purified by flashchromatography to afford the title compound (0.118 g).

The compound obtained in this step has the following mass spectral data:LC/MS: C₃₀H₃₅F₃N₄O₅ requires 588.3; observed M/Z 589.1 [M+H]⁺. RT 8.07min.

Step 8: (3R,5S,E)-ethyl3,5-dihydroxy-7-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoate,41 is prepared as follows:

The product (0.045 g) is obtained in the same manner as Example 10a,Step 8 from ethyl2-((4R,6S)-6-((E)-2-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)vinyl)-2,2-dimethyl-1,3-dioxan-4-yl)acetate,40 (0.118 g).

The compound obtained in this step has the following mass spectral data:LC/MS: C₂₇H₃₁F₃N₄O₅ requires 548.2; observed M/Z 549.1 [M+H]⁺. RT 5.50min.

Step 9:(3R,5S,E)-3,5-dihydroxy-7-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoicacid calcium salt, 42 is prepared as follows:

The product (0.015 g) is obtained in the same manner as Example 10a,Step 9 from (3R,5S,E)-ethyl3,5-dihydroxy-7-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoate,41 (0.045 g).

The compound obtained in this step has the following mass spectral andNMR data:

LC/MS as free acid: C₂₅H₂₇F₃N₄O₅ requires 520.2; observed M/Z 521.0[M+H]⁺, 519.2 [M−H]⁻. RT 2.67 min.

¹H-NMR (270 MHz, CD₃OD, δ) 1.37(d, 6H), 1.38–1.71(m, 2H), 2.18–2.36(m,2H), 3.05–3.09(m, 1H), 3.07(s, 3H), 3.88–3.98(m, 1H), 4.18–4.28(m, 1H),5.65(dd, 1H), 6.24(d, 1H), 7.53–7.76(m, 5H), 8.46(d, 1H).

Example 11f

Synthesis of an N-Pyrimidinyl Pyrazole

In a sixth specific example, a compound having the structure indicatedbelow is prepared in two steps (Steps 1–2) from intermediate 41 obtainedin Example 11e above.

Step 1:(3R,5S,E)-3,5-dihydroxy-7-(3-isopropyl-1′-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoicacid, 43 is prepared as follows:

To a solution of (3R,5S,E)-ethyl3,5-dihydroxy-7-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoate,41 (0.031 g) in ethanol (0.5 mL) is added aqueous sodium hydroxide (57μL of a 1M solution). After stirring at room temperature for 0.25 h,organic solvent is removed in vacuo and aqueous hydrochloric acid (57 μLof a 1M solution) is added, along with water (2 mL). The solution isextracted with ethyl acetate, then washed with brine, dried overmagnesium sulfate and concentrated to afford the title compound (0.024g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₅H₂₇F₃N₄O₅ requires 520.2; observed M/Z 521.0 [M+H]⁺,519.2 [M−H]⁻, RT 2.67 min.

Step 2:(4R,6S,E)-4-hydroxy-6-(2-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)vinyl)-tetrahydropyran-2-one,44 is prepared as follows:

(3R,5S,E)-3,5-dihydroxy-7-(3-isopropyl-1-(2-methoxypyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoicacid, 43 (0.024 g) is dissolved in toluene (3 mL) and heated to 90° C.for 16 h. On cooling, the residue is concentrated in vacuo, thenpurified by flash chromatography to give the title compound (0.010 g).

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS: C₂₅H₂₅F₃N₄O₄ requires 502.2; observed M/Z 503.0 [M+H]⁺, 501.2[M+H]⁻. RT 5.84 min.

¹H-NMR (270 MHz, CDCl₃, δ) 1.37(d, 6H), 1.53–1.95(m, 2H), 2.52–2.77(m,2H), 3.08(s, 3H), 3.07–3.22(m, 1H), 4.30–4.33(m, 1H), 5.08–5.12(m, 1H),5.58(dd, 1H), 6.27(d, 1H), 7.41–7.70(m, 5H), 8.45(d, 1H).

Example 11g

Synthesis of an N-pyrimidinyl Pyrazole

In a seventh specific example, a compound having the structure indicatedbelow is prepared in eight steps (Steps 1–8).

Step 1: 3-oxo-3-(3-(trifluoromethyl)phenyl)propanal, 45 is prepared asfollows:

The product (4.81 g) is obtained as an oil in the same manner as Example10a, Step 1 from 3-(trifluoromethyl)benzaldehyde (10.00 g) and ethylformate (3.94 g)

The compound obtained in this step shows the following boiling point andmass spectral data:

b.p 67–75° C. at 1 mmHg.

LC/MS: C₁₀H₇F₃O₂ requires 216.0; observed M/Z 215.2 [M−H]⁻. RT 7.49 min.

Step 2:2-(methylthio)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,46 is prepared as follows;

The product (2.33 g) is obtained in the same manner as Example 11e, Step2 from 3-oxo-3-(3-(trifluoromethyl)phenyl)propanal, 45 (2.59 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 1.73(s, 3H), 6.52(d, 1H), 7.52–7.55(m, 1H), 7.60 (d,1H), 7.61–7.72(m, 3H), 7.80(s, 1H), 8.55(d, 1H).

Step 3:2-(methylsulfonyl)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,47 is prepared as follows:

The product (2.04 g) is obtained in the same manner as Example 11a, Step3 from2-(methylthio)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,46 (2.33 g). The material is used crude after work-up in the next step.

Step 4:4-(4-bromo-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,48 is prepared as follows:

The product (1.50 g) is obtained in the same manner as Example 10a, Step5 from2-(methylsulfonyl)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,47 (1.84 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₅H₁₀BrF₃N₄O₂S requires 446.0; observed M/Z 446.8/448.8(Br) [M+H]⁺. RT 5.12 min.

Step 5:4-(4-bromo-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-N-phenylpyrimidin-2-amine,49 is prepared as follows:

A solution of4-(4-bromo-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-2-(methylsulfonyl)pyrimidine,48 (0.100 g) and aniline (42 μL) in dimethyl sulfoxide (1 mL) andtrifluoroacetic acid (40 μl) are heated to 90° C. for 16 h. The reactionmixture is then partitioned between ethyl acetate and brine, extractingthe aqueous layer with further ethyl acetate. The combined organiclayers are dried over magnesium sulfate, filtered and concentrated.Purification by flash chromatography affords the title compound (0.06g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₃BrF₃N₅, requires 459.0; observed M/Z 459.9/461.9 (Br)[M+H]⁺, 458.0/460.0 (Br) [M−H]⁻. RT 6.30 min.

Step 6: ethyl2-((4R,6S)-2,2-dimethyl-6-((E)-2-(1-(2-(phenylamino)pyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)vinyl)-1,3-dioxan-4-yl)acetate,50 is prepared as follows:

Bistriphenylphosphine palladium (II) chloride (0.16 g) is added to adegassed solution of4-(4-bromo-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)-N-phenylpyrimidin-2-amine,49 (0.86 g), ethyl2-((4S,6R)-2,2-dimethyl-6-vinyl-1,3-dioxan-4-yl)acetate, 9 (0.72 g) andtriethylamine (3 mL) in DMF (3 mL) and the mixture is heated to 100° C.After 18 h a further portion of bistriphenylphosphine palladium (II)chloride (0.16 g) is added and heating continued. After a further 24 hthe reaction mixture is filtered through celite and evaporated todryness. The residue is partitioned between ethyl acetate and aqueoussodium hydrogen carbonate and the organic layer is washed with water andbrine, evaporated to dryness and purified flash chromatography to affordtitle compound (0.103 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₂H₃₂F₃N₅O₄ requires 607.2; observed M/Z 608.1 [M+H]⁺. RT7.74 min.

Step 7: (3R,5S,E)-ethyl3,5-dihydroxy-7-(1-(2-(phenylamino)pyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoate,51 is prepared as follows:

ethyl2-((4R,6S)-2,2-dimethyl-6-((E)-2-(1-(2-(phenylamino)pyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)vinyl)-1,3-dioxan-4-yl)acetate,50: (0.100 g) and p-tolue sulfonic acid monohydrate (0.033 g) aredissolved in a mixture of tetrahydrofuran (1.5 mL) containing water (0.5mL). After 18 h a further portion of p-toluenesulfonic acid (0.033 g) isadded and stirring continued. After 4 days the reaction mixture ispartitioned between ethyl acetate and aqueous sodium hydrogen carbonate.The organic layer is dried over potassium carbonate and evaporated invacuo to furnish a brown oil. Purification by flash columnchromatography affords the title compound (0.031 g).

The compound obtained in this step shows the following NMR data: LC/MS:C₂₉H₂₈F₃N₅O₄ requires 567.2; observed M/Z 568.0 [M+H]⁺, 566.2 [M−H]⁻. RT5.05 min.

Step 8:(3R,5S,E)-3,5-dihydroxy-7-(1-(2-(phenylamino)pyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoicacid calcium salt, 52 is prepared as follows:

Aqueous sodium hydroxide solution (0.054 mL of a 1 M solution) is addedto a solution of (3R,5S,E)-ethyl3,5-dihydroxy-7-(1-(2-(phenylamino)pyrimidin-4-yl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-4-yl)hept-6-enoate,51 (0.031 g) in ethanol (0.4 mL) and the mixture is stirred at roomtemperature. After 0.5 h the ethanol is removed in vacuo and theremaining aqueous solution is cooled to 0° C. Aqueous calcium chloridesolution (0.23 mL of a 0.118 M solution) is added and the targetmolecule is collected by filtration as a solid.

The compound obtained in this step shows the following mass spectral andNMR data:

LC/MS as free acid: C₂₇H₂₄F₃N₅O₄ requires 539.2; observed M/Z 540.0[M+H]⁺, 538.2 [M−H]⁻. RT 2.79 min.

¹H-NMR (DMSO-d₆, δ) 1.44 (m, 1H), 1.58 (m, 1H), 1.99 (m, 1H), 2.14 (dd,1H) 3.82 (bm, 1H), 4.22 (q, 1H), 6.21 (dd, 1H), 6.30 (dd, 1H), 6.79 (t,1H), 6.86 (bs, 1H), 6.88 (bs, 1H), 6.94–6.98 (m, 2H), 7.23 (d, 1H),7.60–7.76 (m, 4H), 8.27 (s, 1H), 8.56 (d, 1H), 9.56 (bs, 1H).

Example 11h

Synthesis of an N-pyrimidinyl Pyrazole Using an Alternative Route toIntermediate 46 of Example 11g Above.

Intermediate 46 above can also be obtained in two steps (Steps 1–2)detailed below:

Step 1:(E)-3-(dimethylamino)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one, 53is prepared as follows:

A mixture of N,N-dimethylformamide dimethylacetal (2.5 g) and3′-(trifluoromethyl) acetophenone (4.0 g) is heated to 100° C. for 7 h.The crude reaction product (5.22 g) is used without furtherpurification.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₂H₁₂F₃NO requires 243.1; observed M/Z 244.3 [M+H]⁺. RT4.17 min.

Step 2:2-(methylthio)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,46 is prepared as follows:

A solution of(E)-3-(dimethylamino)-1-(3-(trifluoromethyl)phenyl)prop-2-en-1-one, 53(0.50 g) and 4-hydrazinyl-2-(methylthio)pyrimidine, 35 (0.32 g) inacetic acid (5.0 mL) is heated to 80° C. for 12 h. The reaction mixtureis diluted with dichloromethane and washed with saturated aqueous sodiumhydrogen carbonate until the aqueous washings are basic. The organiclayer is dried over magnesium sulfate, filtered and concentrated invacuo to afford an oil. ¹H NMR analysis indicates a 4:1 ratio of isomersin favour of2-(methylthio)-4-(5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,46.

Example 11i

Synthesis of an N-pyrimidinyl Pyrazole

In an eighth specific example, a compound having the structure indicatedbelow is prepared in two steps (Steps 1–2).

Step 1: 4,4,4-trifluoro-1-(3-(trifluoromethyl)methyl)butane-1,3-dione,55 is prepared as follows:

To a flask at 0° C. containing sodium hydride (8.5 g) under nitrogen isadded dropwise a solution of 3′-(trifluoromethyl)acetophenone (20 g) inanhydrous 1,4-dioxane (200 mL). After stirring for 15 min a solution ofethyl trifluoroacetate (15.1 g) in anhydrous 1,4-dioxane (200 mL) isadded dropwise, during which time vigorous gas evolution occurs. Thereaction is left to stir overnight at ambient temperature. The reactionis poured into iced hydrochloric acid solution, then extracted twicewith ethyl acetate. The combined organic layers are washed with brine,dried over magnesium sulfate, filtered and concentrated to a brown oil.The oil is purified by flash chromatography to afford 18 g of the titlecompound as an oil.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₁H₆F₆O₂ requires 284.0; observed M/Z 283.2 [M−H]⁻, RT2.85 min.

Step 2:2-(methylthio)-4-(3-(trifluoromethyl)-5-(3-(trifluoromethyl)phenyl)-1H-pyrazol-1-yl)pyrimidine,56 is prepared as follows:

To a solution of4,4,4-trifluoro-1-(3-(trifluoromethyl)methyl)butane-1,3-dione, 55 (1.00g) in acetic acid (10 mL) is added4-hydrazinyl-2-(methylthio)pyrimidine, 35 (0.55 g) and the reaction isleft to heat to reflux for 48 h. On cooling, the solution is quenchedwith sodium hydrogen carbonate and extracted with ethyl acetate. Theaqueous layer is extracted with a further portion of ethyl acetate andthe combined organic layers dried over magnesium sulfate, filtered andconcentrated to an oil. Flash chromatography affords the title compound(0.11 g) an oil.

The compound obtained in this step shows the following mass spectral andNMR data: LC/MS: C₁₆H₁₀F₆N₄S requires 404.1; observed M/Z 404.9 [M+H]⁺,RT 6.43 min.

¹H NMR (270 MHz, CDCl₃, δ) 1.72 (s, 3H), 6.77 (s, 1H), 7.54 (m, 2H),7.63 ( 1H), 7.69 (m, 2H), 8.60 (d, 1H).

Example 12

Scheme for Synthesis of Pyrrole Compounds

An scheme for synthesizing a substituted pyrrole compounds of formulaVII of the instant invention is provided below. A compound of structure65 can be prepared in nine steps (Steps 1–9) as detailed below.

Step 1: (E)-4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one, 57 isprepared as follows:

N,N-dimethylformamide dimethylacetal (67.21 g) and1,1-dimethoxypropan-2-one (66.62 g), are heated together at 100° C. for16 h. Residual methanol is removed in vacuo and the product used crudein the next step.

Step 2: 4-(dimethoxymethyl)-2-(propylthio)pyrimidine, 58 is prepared asfollows:

(E)-4-(dimethylamino)-1,1-dimethoxybut-3-en-2-one, 57 (10.0 g), andthiourea (4.40 g) are dissolved in methanol (50 mL) at 0° C. and sodiummethoxide (3.12 g) is added portionwise. The mixture is heated to 80° C.for 22 h, then left at ambient temperature for 24 h. 1-Bromopropane(5.25 mL) is added and the mixture warmed to 50° C. for 5 h. The mixtureis concentrated in vacuo and partitioned between ethyl acetate andwater. The aqueous layer is washed with further ethyl acetate and thecombined organics dried (magnesium sulphate), filtered and concentrated.Purification by flash chromatography affords the title compound as anoil (8.53 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₀H₁₆N₂O₂S requires 228.1; observed M/Z 229.2 [M+H]⁺. RT4.75 min.

Step 3: 4-(dimethoxymethyl)-2-(propylsulfonyl)pyrimidine, 59 is preparedas follows:

To a solution of 4-(dimethoxymethyl)-2-(propylthio)pyrimidine, 58 (8.35g) in a mixture of tetrahydrofuran (144 mL) and methanol (320 mL) isadded a suspension of Oxone (90.05 g) and sodium acetate trihydrate(50.00 g) in water (80 mL). The resulting suspension is stirred for 16 hat ambient temperature after which time the organic solvent is removedin vacuo. The residue is partitioned between ethyl acetate and water.The organic layer is washed with aqueous sodium hydrogen carbonate,brine, dried (magnesium sulfate), filtered and concentrated to affordthe title compound (8.66 g) as an oil.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₀H₁₆N₂O₄S requires 260.1; observedd M/Z 261.1 [M+H]⁺. RT3.13 min.

Step 4: 4-(dimethoxymethyl)-2-methoxypyrimidine, 60 is prepared asfollows:

4-(dimethoxymethyl)-2-(propylthio)pyrimidine, 59, is dissolved inmethanol (40 mL) and sodium methoxide (1.57 g) is added. The mixture isheated to 60° C. for 1 h, then the solvent removed in vacuo. The residueis partitioned between ethyl acetate and water, extracting the aqueouslayer with further ethyl acetate. The combined organics are dried(magnesium sulfate), filtered and concentrated to give the titlecompound (4.53 g) as an oil.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₈H₁₂N₂O₃ requires 184.1; observed M/Z 185.2 [M+H]⁺. RT2.57 min.

Step 5: 2-methoxypyrimidine-4-carbaldehyde, 61 is prepared as follows:

4-(dimethoxymethyl)-2-methoxypyrimidine, 60 (1.50 g) is dissolved indilute hydrochloric acid (1M, 12.3 mL) and the mixture heated to 55° C.for 3 h. The reaction mixture is partitioned between water anddichloromethane and the aqueous layer extracted with further portions ofdichloromethane. The combined organics are dried (magnesium sulfate),filtered and concentrated to a yellow gum (1.10 g). The aldehyde is usedimmediately in the next step without further purification.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₆H₆N₂O₂ requires 138.0; observed M/Z 139.1 [M+H]⁺. RT 0.85min.

Step 6:(Z)-2-((2-methoxypyrimidin-4-yl)methylene)-4-methyl-3-oxo-N-phenylpentanamide,62 is prepared as follows:

To a mixture of 2-methoxypyrimidine-4-carbaldehyde, 61 (0.833 g) and4-methyl-3-oxo-N-phenylpentanamide (1.23 g) are added piperidine (4drops) and acetic acid (4 drops). The mixture is heated to 65° C. for 3h after which time it is partitioned between water and dichloromethane.The aqueous layer is extracted with further dichloromethane and thecombined organics dried (magnesium sulfate), filtered and concentrated.Purification by flash chromatography followed by trituration withtoluene affords the title compound (0.221 g) as a powder.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₈H₁₉N₃O₃ requires 325.1; observed M/Z 326.1 [M+H]⁺, 324.2[M−H]⁻. RT 4.02 min.

Step 7:2-(2-(4-fluorophenyl)-1-(2-methoxypyrimidin-4-yl)-2-oxoethyl)-4-methyl-3-oxo-N-phenylpentanamide,63 is prepared as follows:

To a solution of 3-benzyl-5-(2-hydroxyethyl)-4-methylthiazolium chloride(0.166 g) in anhydrous tetrahydrofuran (2 mL) are added(Z)-2-((2-methoxypyrimidin-4-yl)methylene)-4-methyl-3-oxo-N-phenylpentanamide,62 (0.200 g), triethylamine (63 μL) and 4-fluorobenzaldehyde (66 μL).The mixture is heated to 60° C. for 3 h, then partitioned between waterand dichloromethane. The organic layer is dried (magnesium sulfate),filtered and concentrated. The residue is purified by flashchromatography to give the title compound (0.046 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₅H₂₄FN₃O₄ requires 449.2; observed M/Z 450.0 [M+H]⁺,448.2 [M−H]⁻. RT 5.05 min.

Step 8: (3R,5R)-tert-butyl7-(2-(4-fluorophenyl)-5-isopropyl-3-(2-methoxypyrimidin-4-yl)-4-(phenylcarbamoyl)-1H-pyrrol-1-yl)-3,5-dihydroxyheptanoate,64 is prepared as follows:

A mixture of2-(2-(4-fluorophenyl)-1-(2-methoxypyrimidin-4-yl)-2-oxoethyl)-4-methyl-3-oxo-N-phenylpentanamide,63 (0.046 g), tert-butyl2-((4R,6R)-6-(2-aminoethyl)-2-phenyl-1,3,2-dioxaborinan-4-yl)acetate(0.032 g) and pivalic acid (0.010 g) are heated to 80° C. for 16 h. Thesolvent is then removed in vacuo and the residue purified by flashchromatography, during which the boronate moiety is hydrolysed, to givethe title compound (0.024 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₆H₄₃FN₄O₆ requires 646.3; observed M/Z 647.2 [M+H]⁺,645.3 [M−H]⁻. RT 5.24 min.

Step 9:(3R,5R)-7-(2-(4-fluorophenyl)-5-isopropyl-3-(2-methoxypyrimidin-4-yl)-4-(phenylcarbamoyl)-1H-pyrrol-1-yl)-3,5-dihydroxyheptanoicacid calcium salt, 65 is prepared as follows:

To a solution of (3R,5R)-tert-butyl7-(2-(4-fluorophenyl)-5-isopropyl-3-(2-methoxypyrimidin-4-yl)-4-(phenylcarbamoyl)-1H-pyrrol-1-yl)-3,5-dihydroxyhepfanoate,64 (0.019 g) in tetrahydrofuran (0.4 mL) is added aqueous sodiumhydroxide solution (33 μL of a 1 M solution) and the mixture is stirredfor 5 h at ambient temperature. The organic solvent is removed in vacuoand the residue dissolved in water (0.5 mL). Aqueous calcium chloride(265 μL of a 0.118 M solution) is added and the resulting precipitate iscollected by filtration and washed with water and acetonitrile to affordthe title compound (0.003 g).

The compound obtained in this step shows the following mass spectral andNMR data: LC/MS as free acid: C₃₂H₃₅FN₄O₆ requires 590.3; observed M/Z591.1 [M+H]⁺, 589.3 [M−H]⁻. RT 2.62 min.

¹H-NMR (270 MHz, DMSO-d₆, δ) 1.35(d, 6H), 1.53–1.64(m, 2H), 1.81–1.93(m,2H), 1.99–2.11(m, 2H), 3.16(m, 1H), 3.24–3.36(m, 1H), 3.42(s, 3H),3.47–3.57(m, 1H), 3.82–3.93(m, 2H), 4.77(brs, 1H), 6.35(d, 1H), 7.00(t,1H), 7.10–7.15(m, 1H), 7.22–7.43(m, 5H), 7.64(d, 2H), 8.14(d, 1H),10.18(s, 1H).

Example 13

Scheme for Synthesis of Imidazole Cmpounds via Condensation withSidechain

An overall scheme for synthesizing substituted imidazole compouds ofFormula V of the instant invention is provided below. Examples 13a and13b below detail two specific examples following the overall scheme.

Example 13a

Synthesis of Imidazole Cmpound(3R,5R)-7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, Calcium Salt via Condensation with Sidechain

In one specific example,(3R,5R)-7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt, having the structure below, is prepared in foursteps (Steps 1–4) below.

Step 1: N-(pyridin-4-yl(tosyl)methyl)benzamide 66 is prepared asfollows;

To a mixture of pyridine-4-carboxyaldehyde (6.2 g) and benzamide (5.4 g)in 2,2,2-trifluoroethanol (50 mL) is added chlorotrimethylsilane (32.5mL). Upon heating under reflux for 3 h the solvent is evaporated and thefollowing added; acetonitrile (20 mL), toluene (20 mL),4-toluenesulfinic acid (14.0 g) and chlorotrimethylsilane (8.5 mL) inthat order. The suspension is subsequently heated at 55° C. for 4 h,cooled to room temperature and poured slowly. Into a stirred mixture ofsaturated aqueous sodium hydrogen carbonate (300 mL) andtert-butylmethylether (100 mL). After brief agitation, the precipitateis collected and washed with tert-butylmethylether to give the titlecompound, 66 (10.0 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₈N₂O₃S requires 367.1; observed M/Z 228.3[{M−(C₇H₇O₂S)}+NH₃]⁺. RT 2.45 min.

Step 2: N-(2-oxo-2-phenyl-1-(pyridin-4-yl)ethyl)benzamide 67 is preparedas follows:

A flask containing a mixture of N-(pyridin-4-yl(tosyl)methyl)benzamide,66 (10.0 g), and 3,4-dimethyl-5-(2-hydroxyethyl)-thiazolium iodide (1.1g) is flushed with nitrogen for 15 min. Dichloromethane (150 mL) andbenzaldehyde are added and the solution heated to 45° C., thentriethylamine (42 mL) is added. After heating for 16 h, the solution iscooled to room temperature and saturated aqueous sodium hydrogencarbonate is added. The layers are separated, the aqueous phaseextracted with additional dichloromethane, and the combined organiclayers are washed with brine. Drying (magnesium sulfate), filtration,concentration and chromatography furnished the title compound, 67 (1.6g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₆N₂O₂ requires 316.1; observed M/Z 317.1 [M+H]⁺. RT4.05 min.

Step 3: (3R,5R)-tert-butyl7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoate68 is prepared as follows:

To a solution of N-(2-oxo-2-phenyl-1-(pyridin-4-yl)ethyl)benzamide, 67(1.6 g) and acetic acid (1.5 mL) in ethanol (50 mL) is added tert-butyl2-((4R,6R)-6-(2-aminoethyl)-2-phenyl-1,3,2-dioxaborinan-4-yl)acetate(7.9 g). Upon heating under reflux for 16 h, the solution is cooled toroom temperature and the solvent is evaporated. The residue ispartitioned between saturated aqueous sodium hydrogen carbonate anddichloromethane, the layers separated and the organic phase washed withbrine, dried (magnesium sulfate), filtered and concentrated.Purification of the residue via chromatography affords the titlecompound, 68 (0.35 g).

The compound obtained at this step shows the following mass spectraldata: LC/MS: C₃₁H₃₅N₃O₄ requires 513.3; observed M/Z 514.1 [M+H]⁺. RT4.64 min.

Step 4:(3R,5R)-7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt 69 is prepared as follows:

To a 0° C. solution of (3R,5R)-tert-butyl7-(2,5-diphenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoate,68 (100 mg) in tetrahydrofuran (2 mL) is added a 1M solution of sodiumhydroxide (0.19 mL) dropwise and the mixture is stirred at roomtemperature for 16 h. Tetrahydrofuran is removed in vacuo until thesolution becomes turbid, then an aqueous solution of calcium chloride(0.118M, 0.17 mL) added dropwise. The resulting precipitate is filtered,washed with water, acetonitrile, water, acetonitrile and dried in vacuo,affording the title compound, 69 (22 mg).

The compound obtained at this step shows the following mass spectraldata: LC/MS: (as free acid) C₂₇H₂₇N₃O₄ requires 457.2; observed M/Z458.1 [M+H]⁺. RT 2.11 min.

Example 13b

Synthesis of Imidazole Cmpound(3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, Calcium Salt via Condensation with Sidechain

In another specific example,(3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt, having the structure below, is prepared in threesteps (Steps 1–3) below, using an intermediate obtained in Example 13a.

Step 1: N-(2-(4-fluorophenyl)-2-oxo-1-(pyridin-4-yl)ethyl)benzamide 70prepared as follows:

The product,N-(2-(4-fluorophenyl)-2-oxo-1-(pyridin-4-yl)ethyl)benzamide, 0.70 (23mg, is prepared from 66 and obtained in the same manner as Example 13a,Step 2 from 4-fluorobenzaldehyde (37 mg).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₅FN₂O₂ requires 334.1; observed M/Z 335.1 [M+H]⁺. RT4.18 min.

Step 2: (3R,5R)-tert-butyl7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoate71 is prepared as follows:

The product, (3R,5R)-tert-butyl7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoate,71 (5 mg), is obtained in the same manner as Example 13a, Step 3 fromN-(2-(4-fluorophenyl)-2-oxo-1-(pyridin-4-yl)ethyl)benzamide, 70 (23 mg).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₁H₃₄FN₃O₄ requires 531.3; observed M/Z 532.2 [M+H]⁺. RT4.70 min.

Step 3:(3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt 72 is prepared as follows:

The product,((3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid, calcium salt, 72 (1 mg), is obtained in the same manner as Example13a, Step 4 from (3R,5R)-tert-butyl7-(5-(4-fluorophenyl)-2-phenyl-4-(pyridin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoate,71 (5 mg).

The compound obtained in this step shows the following mass spectraldata: LC/MS: (as free acid) C₂₇H₂₆FN₃O₄ requires 475.2; observed M/Z476.0 [M+H]⁺. RT 2.34 min.

Example 14

Scheme for Synthesis of Imidazole Compounds via N-alkylation

An overall scheme for synthesizing substituted imidazole compouds ofFormula V of the instant invention via N-alkylation is provided below.Example 14a provides a scheme for synthesizing the sidechain for use inthe N-alkylatoin scheme. Example 14b details a specific examplefollowing the overall N-alkylation scheme.

Example 14a

Scheme for Synthesis of Sidechain used for Synthesis of ImidazoleCompound via N-alkylation

A sidechain for use in synthesizing imidazole compounds via N-alkylationcan be prepared as follows:

Example 14b

Synthesis of Imidazole Compound (3S,5S),(3R,5R)-6-(5-(4-fluorophenyl)-2-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyhexanoicacid, Calcium Salt via N-alkylation

In one specific example, (3S,5S),(3R,5R)-6-(5-(4-fluorophenyl)-2-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyhexanoicacid, calcium salt, having the structure below, is prepared in ten steps(Steps 1–10) below.

Step 1: 1-(4-fluorophenyl)-2-(2-(methylthio)pyrimidin-4-yl)ethanone 73is prepared as follows:

Under an atmosphere of nitrogen at −78° C., n-BuLi (1.6 M in hexanes,8.4 mL) is added dropwise to a solution of diisopropylamine (2.9 mL) inanhydrous tetrahyrofuran (40 mL). After stirring for 5 min, a solutionof 4-methyl-2-(methylthio)pyrimidine (2.0 g) in anhydroustetrahydrofuran (20 mL) is added and stirring continued for a further 30min at −78° C., whereupon a solution4-fluoro-N-methoxy-N-methylbenzamide (2.8 g) in anhydrous tetrahyrofuran(20 mL) is added. The solution is allowed to warm to room temperatureand then poured into a mixture of ethyl acetate and water.

The layers are separated, the aqueous extracted with ethyl acetate andthe combined organic phases dried over sodium sulfate. Filtration andevaporation of the solvent followed by tituration of the residue with amixture of diethyl ether and hexanes (1:10) furnishes the titlecompound, 73 (3.1 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₃H₁₁FN₂OS requires 262.1; observed M/Z 263.2 [M+H]⁺. RT4.81 min.

Step 2:(Z)-1-(4-fluorophenyl)-2-(hydroxyimino)-2-(2-(methylthio)pyrimidin-4-yl)ethanone74 is prepared as follows:

To a suspension of1-(4-fluorophenyl)-2-(2-(methylthio)pyrimidin-4-yl)ethanone, 73 (1.0 g)in ethanol (20 mL) at −10° C., under an atmosphere of nitrogen, is addeddropwise t-butyl nitrite (0.5 mL) followed by hydrogen chloride inn-propanol (2.5 to 3 N, 0.6 mL) while maintaining the temperature below−5° C. Once the addition is complete, the solution is allowed to warm toroom temperature with stirring and after 2 h the solvent is evaporatedand the residue partitioned between saturated aqueous sodium hydrogencarbonate and ethyl acetate. The layers are separated; the aqueous phaseis extracted with ethyl acetate and the combined organic phases driedover sodium sulfate. Filtration and evaporation of the solvent affordsthe title compound, 74 (1.0 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₃H₁₀FN₃O₂S requires 291.1; observed M/Z 292.0 [M+H]⁺. RT3.74 min.

Step 3:4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)-2-(methylthio)pyrimidine75 is prepared as follows:

A mixture of(Z)-1-(4-fluorophenyl)-2-(hydroxyimino)-2-(2-(methylthio)pyrimidin-4-yl)ethanone(0.5 g), ammonium acetate (2.7 g) and benzaldehyde (0.2 g) in aceticacid (11 mL) are heated under reflux for 4 h, cooled to room temperatureand the majority of the solvent evaporated. The residue is partitionedbetween ice-cold saturated aqueous sodium hydrogen carbonate and ethylacetate. The layers are separated and the aqueous phase is extractedwith ethyl acetate. The combined organic phases are dried over sodiumsulfate, filtered and the solvent evaporated. The residue is dissolvedin methanol (8 mL). This solution is treated with a solution of titanium(III) choride in hydrochloric acid (0.44 mL, 15 wt %, 20–30%hydrochloric acid), followed by stirring at room temperature for 3 h andsolvent evaporation. The residue is partitioned between ice-coldsaturated aqueous sodium hydrogen carbonate and ethyl acetate, then thelayers are separated and the aqueous phase is extracted with ethylacetate. The combined organic phases are dried over sodium sulfate,filtered, the solvent evaporated and the residue purified bychromatography to give the title compound, 75 (0.15 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₅FN₄S requires 262.1; observed M/Z 263.1 [M+H]⁺. RT4.39 min.

Step 4:4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)-2-(methylsulfonyl)pyrimidine76 is prepared as follows;

An aqueous solution (40 mL) of Oxone (10.4 g) is added dropwise to astirred ice-cold solution of4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)-2-(methylthio)pyrimidine,75 (2.1 g) in methanol (40 mL). After stirring for 4 h at roomtemperature, the methanol is evaporated, the solution diluted withsaturated aqueous sodium hydrogen carbonate and extracted with ethylacetate. The combined organic phases are dried over sodium sulphate.Evaporation of the solvent furnishes the title compound, 76 (2.1 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₀H₁₅FN₄O₂S requires 394.1; observed M/Z 395.0 [M+H]⁺. RT4.13 min.

Step 5: 4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)pyrimidine 77 isprepared as follows:

To a suspension of4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)-2-(methylsulfonyl)pyrimidine,76 (0.50 g) in ethanol (20 mL) was added in portions, sodium borohydride(0.28 g). After stirring overnight, hydrochloric acid (20 mL, 0.5 M) isadded and the solution is allowed to stir for 30 min, whereupon, thesolution is neutralized with saturated aqueous sodium hydrogen carbonateand extracted with dichloromethane. The combined organic phases arewashed with water and brine, dried over sodium sulfate, filtered andevaporated. The residue is then subjected to chromatography, furnishingthe title compound, 77 (0.29 g).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₁₉H₁₃FN₄ requires 316.1; observed M/Z 317.1 [M+H]⁺. RT5.32 min.

Step 6: Ethyl 2-((4S,6R),(4R,6S)-2,2-di-tert-butyl-6-vinyl-1,3,2-dioxasilinan-4-yl)acetate 78 isprepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under an atmosphere of nitrogen.Dichlorodi-tert-butylsilane (0.37 g) is added to a solution of silvernitrate (0.59 g) and (3S,5R),(3R,5S)-ethyl 3,5-dihydroxyhept-6-enoate(prepared according to Syn Comm; 24(13), 1833, (1994)) (0.25 g) indimethylformamide (5 mL) at 0° C. After 5 min the solution is allowed towarm to room temperature and stirring is continued for 30 min.Triethylamine (0.41 g) is added and after 10 min the mixture ispartitioned between ethyl acetate and saturated aqueous sodium hydrogencarbonate solution. The aqueous layer is extracted with further portionsof ethyl acetate and the combined organic extracts are dried overmagnesium sulfate, filtered, and concentrated to give the titlecompound, 78 (0.48 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 0.99 (s, 9H), 1.03 (s, 9H), 1.27 (t, 3H) 1.52 (m,1H), 1.78 (dt 1H), 2.40 (dd, 1H), 2.52 (dd, 1H), 4.10–4.25 (overlappingm, 3H), 4.57 (m, 1H), 5.07 (d, 1H), 5.32 (d, 1H), 5.83 (ddd, 1H)

Step 7: Ethyl 2-((4S,6S),(4R,5R)-2,2-di-tert-butyl-6-(2-hydroxyethyl)-1,3,2-dioxasilinan-4-yl)acetate79 is prepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under an atmosphere of nitrogen. A solution of9-borabicyclononane in tetrahydrofuran (18.3 mL of a 0.5 M solution) isadded over 0.5 h to a solution of ethyl 2-((4S,6R),(4R,6S)-2,2-di-tert-butyl-6-vinyl-1,3,2-dioxasilinan-4-yl)acetate, 78(1.0 g) in tetrahydrofuran (10 mL) at 0° C. The reaction mixture is setaside at 4° C. for 64 h then cooled to 0° C. Sodium hydroxide (2.9 mL ofa 3 M solution) and hydrogen peroxide (7.1 mL of a 35% solution inwater) are added simultaneously over 1 h, while the temperature ismaintained below 5° C. The reaction mixture is allowed to warm to roomtemperature over 3 h then partitioned between ethyl acetate and brine.The aqueous layer is extracted with further portions of ethyl acetateand the combined organic extracts are dried over magnesium sulfate,filtered, and concentrated to an oil which is purified by flash columnchromatography to afford the title molecule, 79 (0.41 g).

The compound obtained in this step shows the following NMR data: ¹H-NMR(270 MHz, CDCl₃, δ) 0.98 (s, 9H), 1.03 (s, 9H), 1.26 (t, 3H) 1.65 (m,4H), 2.40 (dd, 1H), 2.52 (dd, 1H), 3.84 (m, 2H), 4.13 (q, 2H), 4.35 (m,1H), 4.52 (m, 1H).

Step 8: Ethyl 2-((4S,6S),(4R,6R)-2,2-di-tert-butyl-6-(2-(methylsulfonyloxy)ethyl)-1,3,2-dioxasilinan-4-yl)acetate80 is prepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under an atmosphere of nitrogen.Diisopropylethylainine (0.23 g) is added to a solution of ethyl2-((4S,6S),(4R,5R)-2,2-di-tert-butyl-6-(2-hydroxyethyl)-1,3,2-dioxasilinan-4-yl)acetate,79 (0.30 g) in dichloromethane (5 mL) at −40° C. After 3 minmethanesulfonyl chloride (0.15 g) is added and the mixture is allowed towarm to room temperature. After 16 h the reaction mixture is washed withsaturated aqueous sodium hydrogen carbonate solution and brine, driedover magnesium sulfate and concentrated in vacuo to afford the titlecompound, 80 (0.16 g) which is used without further purification.

Step 9: Ethyl 2-((4S,6S),(4R,6R)-2,2-di-tert-butyl-6-(2-(5-(4-fluoropheny-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)ethyl)-1,3,2-dioxasilinan-4-yl)acetate81 is prepared as follows:

All glassware is pre-dried in a high temperature oven and the reactionmixture is maintained under an atmosphere of nitrogen. To a solution of4-(4-(4-fluorophenyl)-2-phenyl-1H-imidazol-5-yl)pyrimidine, 77 (162 mg)in anhydrous dimethylformamide (1.6 mL) at −40° C. is added dropwise asolution of potassium hexamethyldisilylamide in toluene (1.03 mL, 0.5M). The solution is allowed to warm to room temperature and subsequentlycooled again to −40° C., whereupon a solution of ethyl 2-((4S,6S),(4R,6R)-2,2-di-tert-butyl-6-(2-(methylsulfonyloxy)ethyl)-1,3,2-dioxasilinan-4-yl)acetate,80 (327 mg) in anhydrous dimethylformamide (1 mL) is added. The solutionis then allowed to warm to room temperature, heated at 85° C. for 20 hand cooled to room temperature. The solution is poured into a mixture ofsaturated aqueous sodium hydrogen carbonate and ethyl acetate, thelayers separated, and the organic layer is washed with three portions ofsaturated aqueous sodium hydrogen carbonate and brine. Drying (magnesiumsulfate), filtration, evaporation of the solvent and chromatography ofthe residue furnishes the title compound as a mixture of enantiomers, 81(37 mg).

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₃₆H₄₅FN₄O₄Si requires 664.3; observed M/Z 645.3 [M+H]⁺. RT8.10 min.

Step 10:(3S,5S),(3R,5R)-7-(5-(4-fluorophenyl)-2-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)-3,5-dihydroxyheptanoicacid 82 is prepared as follows:

To a solution of ethyl 2-((4S,6S),(4R,6R)-2,2-di-tert-butyl-6-(2-(5-(4-fluorophenyl)-2-phenyl-4-(pyrimidin-4-yl)-1H-imidazol-1-yl)ethyl)-1,3,2-dioxasilinan-4-yl)acetate,81 (0.037 g) in anhydrous tetrahydrofuran (0.45 mL) is addedtetrabutylammonium fluoride (1 M solution in tetrahydrofuran, 0.115 mL)and the resulting solution stirred at 50° C. for 1 h, affording thetitle compound.

The compound obtained in this step shows the following mass spectraldata: LC/MS: C₂₆H₂₅FN₄O₄ requires 476.2; observed M/Z 477.0 [M+H]⁺,475.2 [M−H]⁻. RT 2.09 min.

Reported HPLC retention times (RT) provided in the Examples above weredetermined under the following conditions:

Column Waters Xterra MS C18 5 micron, 4.6 mm × 50 mm Particle Size 5micron Dimensions 4.6 mm × 50 mm Solvent A water containing 0.1% v/vaqueous ammonia Solvent B acetonitrile containing 0.1% v/v aqueousammonia Flow rate 1.5 mL/min Initial Conditions 95%A:5%B  Time = 7.0mins  5%A:95%B Time = 7.9 mins  5%A:95%B Time = 8.0 mins 95%A:5%B  Time= 10.0 mins 95%A:5%B  Detection UV at 215 and 254 nm.

Example 15 In Vitro Assays for HMG-CoA Reductase and/or Map KinaseInhibitory Activity Using Specific Pyrazole Compounds Described Herein

Table III below summarizes the results of in vitro assays describedabove for HMG-CoA and/or MAP kinase inhibitory activity of specificpyrazole compounds. Pyrazole compounds obtained according to Examples11a, 11b, 11c, 11d, 11e, 11f, 11g, 10a and 10b were tested as indicated,as the results obtained are provided below.

Compound made in IC50 IC50 IC50 Example # HMG-CoA R^(a) p38α MAPK^(b)Whole cell^(c) 11a 33 nM 11 μM 31 μM l1b 6 μM 21 μM l1e 2 nM 36 μM 30 μM11f >100 μM l6 μM 11g >l00 nM 14 μM 52 μM 10a 3 nM 11 μM 21 μM l0b 6 μM14 μM 11c >100 nM 32 μM 18 μM 11d 8 μM 11 μM ^(a)Concentration ofcompound required to inhibit rat liver HMG-CoA reductase by 50%.^(b)Concentration of compound required to inhibit phosphorylation ofmyelin basic protein by recombinant human p38α MAP kinase by 50%.^(c)Concetration of compound required to inhibit LPS-induced TNF-αrelease from human peripheral blood mononuclear cells by 50%.

The above examples are in no way intended to limit the scope of theinstant invention. Further, it can be appreciated to one of ordinaryskill in the art that many changes and modifications can be made theretowithout departing from the spirit or scope of the appended claims, andsuch changes and modifications are contemplated within the scope of theinstant invention.

1. A compound comprising formula VI:

wherein R₁ is

being 0 or any integer; R₂ is optionally substituted alkyl, aryl, orheteroaryl; R₄ is optionally substituted

and R₅ is optionally substituted aryl or heteroaryl, or a salt thereof,with the proviso that when R₂ is an alkyl from 1–3 carbon atoms,trifluoromethyl, diakylamino where alkyl is 1–4 carbon atoms,pyrrolidino, piperidino, morpholino or piperazino, then R₄ issubstituted.
 2. The compound as recited in claim 1 wherein R₂ is analkyl of from one to four carbon atoms.
 3. The compound as recited inclaim 1 wherein R₂ is dimethylamino.
 4. The compound as recited in claim1 wherein R₂ is 1-methylpropyl.
 5. The compound as recited in claim 1wherein R₂ is isopropyl.
 6. The compound as recited in claim 1 whereinR₂ is phenyl.
 7. The compound as recited in claim 1 wherein R₂ ishydrogen.
 8. The compound as recited in claim 1 wherein R₄ is


9. The compound as recited in claim 1 wherein R₄ is


10. The compound as recited in claim 1 wherein R₄ is


11. The compound as recited in claim 1 wherein R₅ is an optionallysubstituted phenyl group.
 12. The compound as recited in claim 1 whereinR₅ is 3-trifluoromethylphenyl.
 13. The compound as recited in claim 1wherein R₅ is 4-fluorophenyl.
 14. A compound comprising at least onestructure selected from:

salt and/or a solvate thereof.
 15. A compound comprising at least onestructure selected from:

or a salt and/or a solvate thereof.
 16. A compound comprising at leastone structure selected from:

or a salt and/or a solvate thereof.
 17. A compound comprising at leastone structure selected from:

salt or a solvate thereof.
 18. A compound comprising at least onestructure selected from:

or a salt or a solvate thereof.
 19. A compound comprising formula VI:

wherein R₁ is

n being 0 or any integer; R₂ is optionally substituted alkyl, aryl, orheteroaryl; R₄ is substituted

and R₅ is optionally substituted aryl or heteroaryl, or a salt thereof.20. A compound comprising formula VIa

wherein R₁ is

n being 0 or any integer; R₂ is optionally substituted alkyl, aryl, orheteroaryl; the pyrimidinyl ring is optionally substituted; and R₅ isoptionally substituted aryl or heteroaryl, or a salt thereof, with theproviso that when R₂ is an alkyl from 1–3 carbon atoms, trifluoromethyl,diakylamino in which alkyl is 1–4 carbon atoms, pyrrolidino, piperidino,morpholino or piperazino, then the pyrimidinyl ring is substituted. 21.A compound comprising formula VIb:

wherein R₁ is

n being 0 or any integer; R₂ is optionally substituted alkyl, aryl, orheteroaryl; the pyridinyl ring is optionally substituted; and R₅ isoptionally substituted aryl or heteroaryl, or a salt thereof, with theproviso that when R₂ is an alkyl from 1–3 carbon atoms, trifluoromethyl,diakylamino in which alkyl is 1–4 carbon atoms, pyrrolidino, piperidino,morpholino or piperazino, then the pyridinyl ring is substituted.
 22. Apharmaceutical composition comprising an effective amount of at leastone compound as recited in claim 1 with a pharmaceutically acceptablecarrier.